Agents, uses and methods for the treatment of synucleinopathy

ABSTRACT

The invention relates to novel monoclonal anti-alpha-synuclein antibodies. The antibodies can be used for treating a synucleinopathy such as Parkinson&#39;s disease (including idiopathic and inherited forms of Parkinson&#39;s disease), Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer&#39;s disease (LBV), Combined Alzheimer&#39;s and Parkinson disease, pure autonomic failure and multiple system atrophy.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/207,859, filed Jul. 12, 2016 (now U.S. Pat. No. 10,800,836), whichclaims priority to British Patent Application No. GB 1512203.9, filedJul. 13, 2015. The contents of the aforementioned applications arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel class of monoclonal antibodythat specifically binds to alpha-synuclein, as well as to methods ofusing these molecules and their alpha-synuclein binding fragments in thetreatment and diagnosis of synucleinopathies.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 8, 2020, is namedLBJ_001DV8_Sequence_Listing.txt and is 41,524 bytes in size.

BACKGROUND OF THE INVENTION

Synucleinopathies, also known as Lewy body diseases (LBDs), arecharacterized by deposition of intracellular protein aggregates that aremicroscopically visible as Lewy bodies (LBs) and/or Lewy neurites, wherethe protein alpha-synuclein is the major component (Jellinger, MovDisord. 2012 January; 27(1):8-30; McKeith et al., Neurology (1996)47:1113-24). Synucleinopathies include Parkinson's disease (PD)(including idiopathic and inherited forms of Parkinson's disease) andDiffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies(DLB), Lewy body variant of Alzheimer's disease (LBV), CombinedAlzheimer's and Parkinson disease (CAPD), pure autonomic failure (PAF)and multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy,Striatonigral Degeneration and Shy-Drager Syndrome)). Synucleinopathiesfrequently have degeneration of the dopaminergic nigrostriatal system,responsible for the core motor deficits in Parkinsonism (rigidity,bradykinesia, resting tremor), but there is also widespread occurrenceof Lewy bodies and dystrophic Lewy neurites in the central, peripheraland autonomic nervous system and brain regions and other organsassociated with non-motor dysfunctions, such as dementia and autonomicnervous system deficits. Several of the non-motor signs and symptoms arethought to precede motor symptoms in Parkinson's disease and othersynucleinopathies. Such early signs include, for example, REM sleepbehaviour disorder (RBD) and loss of smell and constipation (Mahowald etal., Neurology (2010) 75:488-489). Synucleinopathies continue to be acommon cause for movement disorders and cognitive deterioration in theaging population (Galasko et al., Arch. Neurol. (1994) 51:888-95).

Alpha-synuclein is a member of a family of proteins including beta- andgamma-synuclein and synoretin. Alpha-synuclein is expressed in thenormal state associated with synapses and is believed to play a role inregulating synaptic vesicle release and thereby affecting neuralcommunication, plasticity, learning and memory.

Several studies have implicated alpha-synuclein with a central role inPD pathogenesis. The protein can aggregate to form intracellularinsoluble fibrils in pathological conditions. For example, synucleinaccumulates in LBs (Spillantini et al., Nature (1997) 388:839-40; Takedaet al., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci.Lett. (1997) 239:45-8). Mutations in the alpha-synuclein gene as well asduplications and triplications of the gene co-segregate with rarefamilial forms of parkinsonism (Kruger et al., Nature Gen. (1998)18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-7). Animportant finding has been that alpha-synuclein can be secreted into theextracellular fluid and be present in plasma and cerebrospinal fluid(CSF). Several studies, for example by Pacheco et al. (2015) and others(Pacheco et al J Neurochem. 2015 March; 132(6):731-4; Conway et al.,Proc Natl Acad Sci USA (2000) 97:571-576; Volles et al., J. Biochem.42:7871-7878, 2003) have suggested that extracellular-synuclein plays apathogenic role in the brain. They demonstrated that extracellularalpha-synuclein oligomers possesses neurotoxicity toward brain neuronalplasma membranes. Another intriguing hypothesis based on the data ofsynuclein secretion is that a prion-like spread of alpha-synucleinunderlies the progression of Parkinson's disease and othersynucleinopathies (Lee et al. 2014, Nat Rev Neurol. 2014 February;10(2):92-8; Hansen and Li 2012, Trends Mol Med. 2012 May; 18(5):248-55).These findings have given rise to a hope that extracellular-synucleincould be targeted by immunotherapy (Vekrellis et al. 2011, LancetNeurol. 2011 November; 10(11):1015-25).

Naturally occurring alpha-synuclein auto-antibodies have been shown tobe present in both PD patients and healthy controls (Smith et al. 2012,PLoS One. 2012; 7(12):e52285; Maetzler et al. 2014, PLoS One. 2014 Feb.21; 9(2):e88604, Papachroni et al. 2007 J Neurochem. 2007 May;101(3):749-56 and Woulfe et al. 2002, Neurology. 2002 May 14;58(9):1435-6), sometimes increased levels of auto-antibodies toalpha-synuclein in PD (Gruden et al. 2011, J Neuroimmunol. 2011 April;233(1-2):221-7, Gruden et al. 2012, Neuroimmunomodulation. 2012;19(6):334-42 and Yanamandra 2011, PLoS One. 2011 Apr. 25; 6(4):e18513)or decreased auto-antibodies to alpha-synuclein in PD patients comparedto healthy controls have been reported (Besong-Agbo et al 2013,Neurology. 2013 Jan. 8; 80(2):169-75). The possibility that circulatinganti-alpha-synuclein autoantibodies may serve a protective role withrespect to alpha-synuclein aggregation was suggested very early on afterfinding of the auto-antibodies (Woulfe et al. 2002, Neurology. 2002 May14; 58(9):1435-6).

Over expression of alpha-synuclein in transgenic mice mimics somepathological aspects of Lewy body disease. Several different transgeniclines of mice over-expressing alpha-synuclein have been generated in thelast ten years (described in reviews: Koehler et al 2014, PLoS One. 2013May 31; 8(5):e64649; Fleming and Chesselet, 2006, Behav Pharmacol. 2006September; 17(5-6):383-91; Springer and Kahle 2006, Curr Neurol NeurosciRep. 2006 September; 6(5):432-6). Mouse lines with Thy-1 and PDGF-betapromoters develop motor deficits and cognitive deficits and have beenused to demonstrate a neuroprotective effect of antibodies directedagainst alpha-synuclein in vivo. However, none of the transgenic lineshave robust degeneration of dopaminergic neurons, and often the motorphenotypes are driven by expression in motor neurons, which do notnormally degenerate in Parkinson's disease. Therefore, it is not clearif positive outcome of a potential disease modifying treatment ismediated through effects on dopaminergic neurons or other centralnervous system neurons.

One robust finding in the transgenic mouse models has been that chronicoverexpression of human alpha-synuclein impairs synaptic function. Usingstudies in both in vitro and in vivo systems it was shown thatoverexpression of wild-type (wt) human alpha-synuclein impaired synaptictransmission in hippocampus (Nemani et al. 2010, Neuron. 2010 Jan. 14;65(1):66-79; Paumier et al. 2013, PLoS One. 2013 Aug. 1; 8(8):e70274).This was shown in the CA1 region of the hippocampus where both studiesfound reduced basal synaptic transmission. The mechanism behind this wasassumed to be intracellular accumulation of alpha-synuclein leading todysfunctional synaptic release. However, the recent findings aboutsecretion of alpha-synuclein into extracellular space in synapses andthe toxic effects of alpha-synuclein oligomers on synapse function opensfor the possibility of a role of extracellular alpha-synuclein insynaptic dysfunction, and as such for the ability of therapeuticantibodies to rescue the deficit.

The use of viral vectors to over-express alpha-synuclein represents animportant way to model PD in rodents because this approach produces arelative fast progressive degeneration of nigrostriatal neurons, afeature not yet reproduced by genetic mutations in mice or rats (Kirikand Bjorklund, 2003, Trends Neurosci. 2003 July; 26(7):386-92).Furthermore, viral gene delivery revealed the ability of wtalpha-synuclein to induce nigrostriatal pathology (Kirik et al. 2002, JNeurosci. 2002 Apr. 1; 22(7):2780-91), a finding in agreement withevidence in familial forms of PD with alpha-synuclein dublications andtriplications (Lee and Trojanowski, 2006, Neuron. 2006 Oct. 5;52(1):33-8). In one study, it has been shown that a a pool of goatantibodies against the alpha-synuclein N-terminal protected againstdopaminergic cell death and ameliorated behavioural deficits in aAAV-alpha-synuclein based rat model of Parkinson's disease(Shahaduzzaman et al 2015, PLoS One. 2015 Feb. 6; 10(2):e0116841).

Prion like spreading of alpha-synuclein pathology has recently beenshown to develop alpha-synuclein pathology and also develop dopaminergiccell death (Luk et al. 2012, Science. 2012 Nov. 16; 338(6109):949-53).This model has been used to show that alpha-synuclein antibodies areable to ameliorate the pathology (Tran et al. 2014, Cell Rep. 2014 Jun.26; 7(6):2054-65). In this model antibody treatment was able to reduceaccumulation of phosphorylated alpha-synuclein in several brainregions—including dopaminergic neurons in substantia nigra, and reducedevelopment of motor deficit.

In addition to mutations, alternative splicing of the alpha-synucleingene and posttranslational modifications of the protein, such asphosphorylation, ubiquitination, nitration, and truncation can createalpha-synuclein protein forms that have enhanced capacity to formaggregated and/or toxic forms of alpha-synuclein (Beyer and Ariza, MolNeurobiol. 2013 April; 47(2):509-24). However, the precise pathologicalspecies of alpha-synuclein remains unknown. Variousmisfolded/aggregated/secreted species ranging from oligomers to fibrils,and different post-translational modifications have been associated withtoxicity but there is no consensus on which is most important, if indeedthere even is a single toxic species.

Overall the accumulation of alpha-synuclein with similar morphologicaland neurological alterations in animal models as diverse as humans,mice, and flies suggests that this molecule is central in thepathogenesis of Lewy body diseases.

Several different antibodies to alpha-synuclein have been shown to havetherapeutic effect in preclinical animal models. Both an antibodytargeting an epitope involving alpha-synuclein residues 91-99 andantibodies targeting an epitope that involves alpha-synuclein residues118-126 have been shown to have an effect on motor and cognitivedeficits in transgenic mice (Games et al. 2014, J Neurosci. 2014 Jul. 9;34(28):9441-54). The most advanced of these antibodies is a humanizedantibody based on the mouse monoclonal antibody 9E4, which targets anepitope that involves alpha-synuclein residues 118-126, and which is nowin clinical trials in phase I. A C-terminal antibody 274 which targetsan epitope that involves alpha-synuclein residues 120-140 (Bae et al.2012, J Neurosci. 2012 Sep. 26; 32(39):13454-69) was also shown to havean effect in a preclinical model on spreading of the pathology from cellto cell. In addition to these, antibodies targeting conformationalspecies such as oligomers and fibrils of alpha-synuclein have been shownto be able to at least reduce the levels of these presumably toxicalpha-synuclein species (Lindstrom et al. 2014, Neurobiol Dis. 2014September; 69:134-43 and Spencer et al. 2014, Mol Ther. 2014 October;22(10):1753-67). These conformational antibodies that loweralpha-synuclein oligomer levels in vivo, such as mab47 were also shownto target epitopes in the C-terminus of alpha-synuclein, from amino acid121-125 (US20120308572). Other conformational, fibril and oligomerspecific antibodies also target C-terminal sequences (Vaikath et al.Neurobiol Dis. 2015; 79:81-99).

As the toxic form of alpha-synuclein is unknown, a therapeutic antibodyshould be ideally able to bind to most of the alpha-synuclein speciesthat are formed by alternative splicing or posttranslationalmodifications, such as truncations, as well as oligomeric and fibrillaryforms. One problem with current antibodies that have been tested astherapeutics in preclinical models, as discussed above, is that many ofthem target C-terminal epitopes, which are not found in some of themajor truncated forms of alpha-synuclein. For example, the amino acidsthat are important for binding of 9E4 are asparagine 122 and tyrosine125 (according to an alanine scan presented in patent US20140127131),and this means that this antibody cannot bind alpha-synuclein which istruncated at amino acids 119, and 122, which are some of the majortruncated species in Parkinson brain tissue (Kellie et al. Sci Rep.2014; 4:5797). The same would be the case for the antibody 274 andantibody mab47 (U.S. Pat. No. 8,632,776). Also, amino terminalantibodies would possibly not be able to bind to some of the majortruncated species that lack the first amino acids of alpha-synuclein,such as alpha-synuclein truncated to amino acids 5-140. For the 9E4antibody, one suggested mechanism of action is the prevention oftruncation at amino acids 119-122 in extracellular space, as theantibody will bind to the same region where the protease that willcleave alpha-synuclein (Games et al. 2014, J Neurosci. 2014 Jul. 9;34(28):9441-54). A similar mechanism of action could also be found withantibodies in close proximity of the site, and therefore many antibodiesaround this region would be expected to have this activity.

There is some support for a toxic role of the truncated alpha-synucleinspecies in animal models. Expression of truncated alpha-synuclein underthe tyrosine-hydroxylase promoter has been shown to lead tonigrostriatal pathology, which is normally not seen in transgenicalpha-synuclein models (Tofaris et al. 2006, J Neurosci. 2006 Apr. 12;26(15):3942-50; Wakamatsu et al. 2006, Neurobiol Aging. 2008 April;29(4):574-85). For example, expression of amino acids 1-130 of a humanalpha-synuclein protein having the A53T mutation caused embryonic lossof dopaminergic neurons in the substantia nigra pars compacta whereasexpression of the full length protein did not (Wakamatsu et al. 2006,Neurobiol Aging. 2008 April; 29(4):574-85). Expression of a 120 aminoacid alpha-synuclein molecule under the calcium/calmodulin-dependentprotein kinase II alpha (CamKII-alpha) promoter was associated withalpha-synuclein aggregation and a progressive deficit incortical-hippocampal memory tests including the Barnes maze and novelobject recognition (Hall et al. 2015, Exp Neurol. 2015 February;264:8-13). Also in the rat AAV model co-expression of C-terminaltruncated alpha-synuclein enhanced full-length alpha-synuclein-inducedpathology (Ulusoy et al. 2010, Eur J Neurosci. 2010 August;32(3):409-22).

In this invention, antibodies (such as “GM37” and “GM285”, described inthe Examples) have been generated that can bind to the toxicalpha-synuclein fragment 1-119/122 and neutralize this truncated form ofalpha-synuclein. The antibodies of the invention, such as GM37 andGM285, are capable of binding to other oligomeric forms ofalpha-synuclein and altering their uptake by other CNS resident cells ina manner that reduce the spreading of disease. Furthermore, theantibodies of the invention, such as GM37 and 285, were surprisinglyfound to be superior to prior art antibodies such as 9E4 in binding todifferent alpha-synuclein species in human brain, and has a surprisingsuperior effect on clearing extracellular alpha-synuclein andnormalising impaired synaptic transmission induced by the presence ofabnormal alpha-synuclein in vivo. Further illustrating their therapeuticcapabilities, the antibodies of the invention, such as GM37 and 285, areable to prevent the appearance of a disease related motor phenotype in arat model for Parkinson's disease. Finally, antibodies GM37 and GM285are able to inhibit seeding of aggregation and phosphorylation ofendogenous alpha-synuclein induced by extracellular added recombinantpathological alpha-synuclein seeds in primary mouse neurons. Antibodiessuch as GM37 and 285 can also inhibit seeding of alpha-synucleinpathology into dopaminergic neurons in vivo using a mouse model forParkinson's disease, further supporting the therapeutic capability ofthese antibodies in preventing the cell to cell propagation ofpathology. Together these data strongly support the use of these novelantibodies, GM37 and GM285, as new therapeutic agents capable ofmodifying disease through inhibition of the mechanism by which thedisease pathology spreads between the neurons Parkinson's patients.

In a further aspect of the invention is provided 3 amino acid variantsof the GM37 antibody. All the variants have similar functional readoutsas the parent antibody, GM37, but with improved properties formanufacturability. The variants reduce the risk of post-translationalmodification occurring within the binding domain of the GM37 antibodyand provide some improvement in the production of the antibody. This isadvantageous because large scale clinical or commercial manufacturing ofantibodies is complicated and expensive, and providing a homogenousproduct in pharmaceutical medicaments is crucial in particular forimmunoglobulins and proteins.

SUMMARY OF THE INVENTION

The invention relates to novel monoclonal antibodies, andantigen-binding fragments thereof, capable of specifically binding anepitope within amino acids 112-117 in alpha-synuclein (SEQ ID NO:9(ILEDMP)). The epitope bound by the antibodies or antibody-bindingfragments thereof of the invention, such as exemplary antibody “GM37”,or “GM285”, is referred to herein as “the 112-117 epitope”. Theantibodies of the present invention specifically bind to an epitopewithin the 112-117 epitope and may, according to one embodiment, competewith antibody GM37 or GM285 for binding to an epitope within amino acids112-117. For example, antibodies or antigen-binding fragments thereofaccording to the invention may compete for binding to an epitope withinamino acids 112-117 of human alpha-synuclein with a heavy chainconsisting of a variable domain of SEQ ID NO:7 and a light chainconsisting of a variable domain of SEQ ID NO:8. Such competitive bindinginhibition can be determined using assays and methods well known in theart, for example using an unlabelled binding assay such as surfaceplasmon resonance (SPR). For example, immobilising human alpha-synucleinon a surface and incubating with or without the reference antibody‘GM37’ prior to incubation with an antibody or binding fragment to betested. Alternatively, a pair-wise mapping approach can be used, inwhich the reference antibody ‘GM37’ is immobilised to the surface, humanalpha-synuclein antigen is bound to the immobilised antibody, and then asecond antibody is tested for simultaneous binding ability to humanalpha-synuclein (see ‘BIAcore® Assay Handbook’, GE Healthcare LifeSciences, 29-0194-00 AA 05/2012; the disclosures of which areincorporated herein by reference).

More specifically the GM285 antibody binds an epitope within residues112-117 of alpha-synuclein comprising residues 112-115 ofalpha-synuclein (ILED; SEQ ID NO:19).

In one embodiment, the invention relates to monoclonal antibody GM37,its variants (e.g., GM37 Variant 1, GM37 Variant 2 and GM37 Variant 3),or GM285.

In particular, the invention provides a monoclonal antibody GM37, itsvariants (e.g., GM37 Variant 1, GM37 Variant 2 and GM37 Variant 3), orGM285, and encompasses such antibodies as well as derivatives thereofthat possess a sufficient number (e.g., 1, 2, or 3) light chain CDRs anda sufficient number (e.g., 1, 2, or 3) heavy chain CDRs to form abinding site capable of specifically binding to human synuclein.Preferably, such antibodies will possess the three light chain CDRs andthree heavy chain CDRs, as defined below. The numbering of amino acidresidues in this region is according to IMGT®, the internationalImMunoGeneTics information System® or, Kabat, E. A., Wu, T. T., Perry,H. M., Gottesmann, K. S. & Foeller, C. (1991). Sequences of Proteins ofImmunological Interest, 5th edit., NIH Publication no. 91-3242 U.S.Department of Health and Human Services; Chothia, C. & Lesk, A. M.(1987). Canonical structures For The Hypervariable domains OfImmunoglobulins. J. Mol. Biol. 196, 901-917.

In one embodiment, the monoclonal antibody or antigen-binding fragmentsthereof possesses a synuclein antigen-binding fragment comprising orconsisting of:

-   -   (a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:1; and/or    -   (b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:2; and/or    -   (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:3; and/or    -   (d) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:4; and/or    -   (e) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:5; and/or    -   (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:6; that is capable of specifically binding to human        alpha-synuclein.

In another embodiment, the monoclonal antibody or antigen-bindingfragments thereof possesses a synuclein antigen-binding fragmentcomprising or consisting of:

-   -   (a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:1;    -   (b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:33, 34 or 35;    -   (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:3;    -   (d) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:4;    -   (e) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:5; and    -   (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:6; that is capable of specifically binding to human        alpha-synuclein.

In yet another embodiment, the monoclonal antibody or antigen-bindingfragments thereof possesses a synuclein antigen-binding fragmentcomprising or consisting of:

-   -   (a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:20; and/or    -   (b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:21; and/or    -   (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:22; and/or    -   (d) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:23; and/or    -   (e) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:24; and/or    -   (f) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:25. that is capable of specifically binding to human        alpha-synuclein.

In one embodiment, the monoclonal antibody or antigen-binding fragmentsthereof possesses a synuclein antigen-binding fragment comprising anamino acid sequence (in its CDRs, its variable domains, its frameworkresidues or in its constant domains) that differs from that of naturallyoccurring anti-alpha-synuclein antibodies, and that exhibits (relativeto such naturally occurring anti-alpha-synuclein antibodies):

-   -   (i) a difference in binding affinity (KD) for alpha-synuclein;    -   (ii) a difference in the capability of inhibiting protease        truncation of alpha-synuclein fibrils;    -   (iii) a difference in the capability of reversing impairment in        basal synaptic transmission in F28-snca transgenic mice;    -   (iv) a difference in the capability of reducing levels of        alpha-synuclein in the mouse hippocampus as measured by in vivo        microdialysis; and/or    -   (v) a difference in the capability, when administered        chronically, to restore motor function in a rat model of        Parkinson's disease    -   (vi) a difference in the ability to prevent seeding of        alpha-synuclein (such as accumulation of insoluble        phosphorylated alpha-synuclein in vitro and/or in a mouse model        of Parkinson's disease); and/or    -   (vii) a difference in the capability to bind truncated        alpha-synuclein in a human brain.

The antibodies and antigen-binding fragments thereof of the inventionmay be used in a method to treat, diagnose or image synucleinopathies,such as Parkinson's disease ((PD), including idiopathic and inheritedforms of Parkinson's disease), Diffuse Lewy Body Disease (DLBD), Lewybody variant of Alzheimer's disease (LBV), Gauchers Disease (GD),Combined Alzheimer's and Parkinson disease (CAPD), pure autonomicfailure and multiple system atrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunization protocols for generation of hybridomas. Thetable outlines the differences of the immunogens and mouse strains usedfor the identification of GM37 and GM285. Different HCo17-Balb/c andHCo12/Balb/c mice were immunized independently (description of thesemice are provided below). The hybridoma expressing GM37 was identifiedfrom mice immunized with full length alpha-synuclein containing aminoacids 1-140 fibrils and boosted with truncated alpha-synuclein fragments1-60 and 1-119 of full length (FL) alpha-synuclein (SEQ ID NO 10). Thehybridoma expressing antibody GM285 came from an immunization protocolin which HCo12-Balb/c mice were immunized with full length monomericalpha-synuclein, amino acids 1-140 followed by a boost with full lengthfibrillary alpha-synuclein (Example 1).

FIGS. 2A-2C show screening of GM37 for binding to alpha synuclein,alpha-synuclein homologs and orthologs.

FIG. 2A) Binding of antibody GM37 to alpha-synuclein using a no washsolution based ELISA (FMAT).

FIG. 2B) Using SPR (Fortebio) binding of antibody GM37 is specific foralpha-synuclein (Alpha Panel) and does not bind the other relatedsynuclein family proteins, beta-synuclein (Beta Panel) andgamma-synuclein (Gamma Panel). Measurements were performed using SPR(Fortebio Octetred) GM37 shows similar binding to alpha-synuclein fromcynomolgus monkey (Cyno Panel) and mouse (Mouse Panel). (Example 1).

FIG. 2C) Using SPR (Fortebio Octetred) binding of antibody GM285 isspecific for alpha-synuclein and does not bind the other relatedsynuclein family proteins, beta-synuclein and gamma-synuclein.Measurements were performed using SPR (Fortebio Octetred) shows similarbinding of GM285 to alpha-synuclein from cynomolgus monkey (Cyno) andmouse (Mouse)(Example 1).

FIGS. 3A-3C show real time binding Affinity of GM37

FIG. 3A) Binding of antibody GM37 to alpha-synuclein measured in RU(Relative Units) (y-axis) over time (X-axis) as determined by SPR(BIAcore® 3000). Goat anti-human IgG was immobilized on the CM5 chip.GM37 was captured on the Goat anti-human IgG immobilized chip and seriesof concentrations of human alpha-synuclein (3.125, 6.25, 12.5, 25, 50,100 nM) were tested on binding to the surface. The sensor surface wasregenerated between each cycle.

FIG. 3B) Signal from binding at different concentrations converted intoa binding curve.

FIG. 3C) Calculated binding constants of antibody GM37 (denotedhIgG1-6004-037-C106S) (Example 2).

FIGS. 4A-4C show real time binding Affinity of GM285

FIG. 4A) Binding of antibody GM285 to alpha-synuclein measured in RU(y-axis) over time (X-axis) as determined by SPR (BIAcore® 3000). Goatanti-human IgG was immobilized on the CM5 chip. GM285 was captured onthe Goat anti-human IgG immobilized chip and series of concentrations ofhuman alpha-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested onbinding to the surface. The sensor surface was regenerated between eachcycle.

FIG. 4B) Signal from binding at different concentrations converted intoa binding curve.

FIG. 4C) Calculated binding constants of antibody GM285 (denotedhIgG1-6004-285) (Example 2).

FIGS. 5A-5C show real time binding of comparator antibody 9E4

FIG. 5A) Shows binding of 9E4 to alpha-synuclein measured in RU (y-axis)over time (X-axis) as determined by SPR (BIAcore® 3000). Goat anti-humanIgG was immobilized on the CM5 chip. 9E4 was captured on the chip by itsbinding to Goat anti-human IgG that had been immobilized to the chip. Aseries of concentrations of human alpha-synuclein (3.125, 6.25, 12.5,25, 50, 100 nM) were tested for binding to the surface. The sensorsurface was regenerated between each cycle.

FIG. 5B) Signal from binding at different concentrations converted intoa binding curve.

FIG. 5C) Calculated binding constants for antibody 9E4. (Example 2).

FIG. 6 shows the amino acid sequence of alpha-synuclein. Majortruncation sites (indicated by arrows) identified by mass spectrometryin human brain tissue (Kellie J F, Higgs R E, Ryder J W, Major A, BeachT G, Adler C H, Merchant K, Knierman Md. Quantitative measurement ofintact alpha-synuclein proteoforms from post-mortem control andParkinson's disease brain tissue by mass spectrometry. Sci Rep. 2014Jul. 23; 4:5797. doi: 10.1038/srep05797)

FIGS. 7A and 7B show epitope mapping of antibody GM37 and GM285. ELISAdata showing relative levels of binding of the antibodies to sequentialpeptides (20mers) derived from alpha-synuclein amino acid sequence95-132 (the other nonbinding peptides are not shown).

FIG. 7A) GM37 epitope requires peptide sequence ILEDMP (SEQ ID NO:9) forfull binding.

FIG. 7B) GM285 requires peptide ILED (SEQ ID NO:19) for full binding.(Example 3).

FIGS. 8A and 8B show a schematic representation of truncated forms ofalpha-synuclein.

FIG. 8A) binding epitopes of GM37/285 (ILEDMP; SEQ ID NO:9) and 9E4(NEAYE; SEQ ID NO:36) are shown in bold on the alpha-synuclein aminoacid sequence (SEQ ID NO:10). Arrows indicates the c-terminaltruncations sites from FIG. 6 .

FIG. 8B) Major truncated forms of alpha-synuclein that have beenidentified from human brain material. Size based on amino acid numbersis indicated on the right side. Full length alpha-synuclein is 140 aminoacids. As can be deducted from the epitopes, GM37, it's variants 1-3,and GM285 should bind full length and the 1-119/122, 1-135 fragments.Antibody 9E4 will bind only to full length and 1-135 fragment. Thespecific nature of the smaller c-terminal fragments left after thetruncations are not shown.

FIG. 9 shows that antibodies GM37 and GM285 immunoprecipitate fulllength alpha-synuclein as well as truncated alpha-synuclein from humanbrain. Crude homogenates of human DLB brain were incubated with the testantibodies (Beads (No ab), B12-human IgG1 control antibody not bindingto alpha-synuclein, GM-37, GM37 variant 2, GM-285 and murine (m)9E4) andthe immunodepleted supernatant and immunoprecipitated material wasseparated on SDS-PAGE. The western blot shows the bands representing thefull length and the different truncated forms of alpha-synuclein beingdepleted from the supernatant and being immunoprecipitated with theantibodies (IP). As can be seen, the GM37, GM37v2 and GM285 antibodydepleted the major alpha-synuclein species from the supernatant, and theIP shows these species, the truncated species 1-135, 1-119/122 and fulllength alpha synuclein. The 9E4 does not affect the 1-119/122 speciesbut only IPs full length and 1-135 (Example 4).

FIG. 10 shows schematics of the proteolysis of alpha-synuclein fibrilscleaved by calpain at amino acid 119/122. Alpha-synuclein fibrils (PFF)are added to the culture with (PFF+) or without (PFF) test antibody. Thepresence of GM-37/285 inhibit the formation of the truncatedalpha-synuclein in cells and secreted into the cell media.

FIG. 11A shows that GM37 inhibits the formation of the truncated band(12 KD) in both the media and in cell lysates of primary mouse corticalcultures treated with PFFs. Proteins were separated by SDS-PAGE andwestern blotted to detect different species of alpha-synuclein. In cellstreated only with PFF or the control antibody (B12) two monomericalpha-synuclein bands are detected at 12 and 14 kDa, representingtruncated and full length alpha-synuclein, respectively. In the presenceof GM-37 there is only a faint band at 12 Kd indicating that themajority of the cleavage is blocked. This effect is also reflected inthe in the media of the cells. The relative levels of accumulation mayalso be inhibited by the presence of GM-37 as reflected in the reductionin the relative intensity of the 14 Kd band. Alternatively there may bereduced amount of the 14 Kd band available for uptake by the cells.(Example 5).

FIG. 11B shows dose dependent inhibition of proteolysis ofalpha-synuclein fibrils by antibodies GM37, GM37 variant 2 and GM285. Incell lysates from primary mouse cortical cultures at low antibodyconcentration (0.1 ug/ml) there are both a band representing full length(FL) alpha-synulcein and a band representing C-terminally truncated (CT)alpha-synuclein (indicated by arrows). Increasing antibody concentrationto 1, 5 and 10 ug/ml leads to reduced proteolysis of alpha-synuclienfibrils in cells. This is observed with both antibody GM37, GM37v2 andGM285. Control samples are treated with a human IgG1 antibody B12 notrecognising alpha-synuclein. There is also a control with no antibodyadded (No ab), and cells with no alpha-synuclein fibrils added (NoAsyn). The total amount of alpha-synuclein is also reduced in samplestreated with 37, 37v2 and 285 compared to B12 or “no antibody” control,indicative that all three antibodies reduce accumulation ofalpha-synuclein in cells in concentration dependent manner. The actinband on the top of the gel shows equal loading of the samples (Example5).

FIGS. 12A-12D show the impact of GM37 and GM285 on seeding ofalpha-synuclein aggregation and alpha-synuclein phosphorylation in mouseprimary cortical neurons.

FIG. 12A) Example of images of primary neurons stained forphosphorylated alpha-synuclein, which appears as spots or punctatestaining in cells when the cells are seeded with either 1 ng of pureseeds or crude seeds of alpha-synuclein.

FIG. 12B) Western blot of proteins from primary cortical neuronsseparated in soluble and insoluble fractions. The blots were stainedwith human alpha-synuclein specific antibody (41312/H a-syn),phospho-Ser-129-alpha-synuclein specific antibody (ab51253/pS-a-Syn) andmouse alpha-synuclein specific antibody (D37A2/M a-syn) and show thataddition of the crude seeds in primary neurons leads to accumulation ofendogenous mouse alpha-synuclein and phosphorylated alpha-synuclein andhigher molecular weight multimers of alpha-synuclein in the insolublefraction.

FIG. 12C) GM37, GM37 variant 2 and GM285 inhibit appearance ofphosphorylated alpha-synuclein quantitated as the number ofalpha-synuclein phos-phoserine 129 positive spots in cells by aCellomics ARRAYSCANTMautomated microscope. GM37, GM37v2 and GM285 reducethe amount of phosphorylated alpha-synuclein spots in cells in dosedependent manner.

FIG. 12D) Western blot of the homogenates from primary cortical neuronstreated at the highest dose of antibody (133 nM), and stained for actin,human alpha-synuclein, phosphorylated alpha-synuclein and mousealpha-synuclein shows that antibodies 37, 37v2 and 285 inhibittruncation of the alpha-synuclein crude seeds taken up by the cells inthe insoluble fraction. All antibodies also inhibit the accumulation ofphosphorylated, endogenous mouse and higher molecular weight multimersof phosphorylated mouse alpha-synuclein in the insoluble fraction. Theactin band on the top of the gel shows equal loading of the samples(Example 6).

FIG. 13 shows basal synaptic transmission at the Schaffer collateral-CA1synapse in the hippocampus of F28-snca transgenic and age-matchedcontrol mice. Field excitatory post-synaptic potentials (fEPSPs) wereevoked by a single stimulus applied to the Schaffer collateral, andbasal synaptic transmission was assessed by measuring the fEPSP slope asa function of the stimulation intensity. Short-term synaptic plasticitywas evaluated by induction of paired-pulse facilitation. The differentintensities of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400,and 500 pA, and were applied successively in increasing order, with 2 to3 repeats for each intensity. Basal synaptic transmission was found tobe significantly impaired in F28-snca transgenic mice overexpressingwild-type alpha-synuclein compared to age-matched control mice (Example7).

FIG. 14 shows the effect of the systemic administration of a single doseof human 9E4 (15 mg/kg, i.p.) on the impairment in basal synaptictransmission at the Schaffer collateral-CA1 synapse in the hippocampusof F28-snca transgenic mice. Field excitatory post-synaptic potentials(fEPSPs) were evoked by a single stimulus applied to the Schaffercollateral, and basal synaptic transmission was assessed by measuringthe fEPSP slope as a function of the stimulation intensity. Acutetreatment with h9E4 induced a significant reversal of the impairment inbasal synaptic transmission in F28-snca transgenic mice (Tg-snca+h9E4vs. Tg-snca+PBS, p=0.002). However, the reversal by h9E4 was onlypartial, as indicated by a significantly lower basal synaptictransmission compared to littermates treated with PBS (p=0.007) (Example7).

FIG. 15 shows the effect of the systemic administration of a single doseof human GM37 (15 mg/kg, i.p) or an isotype control antibody (B12) onthe impairment in basal synaptic transmission at the Schaffercollateral-CA1 synapse in the hippocampus of F28-snca transgenic mice.Field excitatory post-synaptic potentials (fEPSPs) were evoked by asingle stimulus applied to the Schaffer collateral, and basal synaptictransmission was assessed by measuring the fEPSP slope as a function ofthe stimulation intensity. Acute treatment with GM37 induced fullreversal of the impairment in basal synaptic transmission in F28-sncatransgenic mice (Tg-snca+GM37 vs. Tg-snca+B12, p=0.004) (Example 7).

FIG. 16 shows the effect of the systemic administration of a single doseof human GM285 (15 mg/kg, i.p) on the impairments in basal synaptictransmission at the Schaffer collateral-CA1 synapse in the hippocampusof F28-snca transgenic mice. Field excitatory post-synaptic potentials(fEPSPs) were evoked by a single stimulus applied to the Schaffercollateral, and basal synaptic transmission was assessed by measuringthe fEPSP slope as a function of the stimulation intensity. Acutetreatment with GM285 induced full reversal of the impairment in basalsynaptic transmission in F28-snca transgenic mice (Tg-snca+GM285 vs.Tg-snca+PBS, p=0.001) (Example 7).

FIGS. 17A and 17B show the effect of the systemic administration (15mg/kg, i.p.) of human 9E4, GM37 or isotype control antibody (anti-HEL)on the levels of human alpha-synuclein in the interstitial fluid (isf)in the hippocampus of freely moving F28-snca transgenic mice. Theaverage of the two-three basal values (4 h-6 h) prior to antibodytreatment was taken as baseline and set to 100% for each animal.Differences were analyzed using a two-way analysis of variance (ANOVA)with repeated measures. The basal levels of human alpha-synuclein inhippocampus were 8.1±1.1 ng/ml (mean±SEM, n=25, not corrected for the invitro dialysis probe recovery). The administration of GM37 induced alarger reduction in human alpha-synuclein in the hippocampus of F28 micecompared to both the comparator antibody, human 9E4, and the controlisotype, anti-HEL Timepoints that show significant differences in thelevels of alpha-synuclein between animals treated with GM37 or thecontrol antibody are indicated by an asterisk. (Example 8).

FIG. 18 shows a schematic representation of the timeline for antibodytreatment (down arrows), viral injections and behavioural assessment inthe rat AAV human alpha-synuclein model shown in FIG. 19 (Example 9).

FIG. 19 shows that antibody GM37 can reduce Parkinsonian motor deficitsafter chronic treatment in the rat AAV model. The effect of chronictreatment with GM37 or PBS in AAV-human-alpha-synuclein rats on motorasymmetry is assessed in the cylinder test. Each rat was tested for theuse of the forepaws by monitoring for 5 minutes. The percentage of useof the right forepaw (ipsilateral to the injection) and use of left(contralateral+right forepaws) was calculated for each animal (as shownon the y-axis) *, ** p<0.05 and 0.01 compared to GFP-PBS rats. The ratstreated with PBS still have a significant asymmetry in paw use, whileanimals treated with antibody GM37 have no longer a significant deficit.(Example 9).

FIGS. 20A-20C show that chronic treatment with antibody GM37 can reducepathological alpha-synuclein phosphorylation induced by injection ofpathological alpha-synuclein fibrillary seeds into the mouse striatum.FIG. 20A shows a schematic indicating relative treatment times withrespect to seed injection and cell counting. The antibody GM37 wasadministered one day before injection of recombinant alpha-synucleinfibrillary seeds into dorsal striatum of mice, and then weekly for sixweeks. Dosing regimen was either 15 mg/kg iv or 30 mg/kg ip. FIG. 20Bshows the exposure level of GM37 in plasma based on site of injectionand dose. Weekly samples were taken before the injection of new antibodydose. FIG. 20C shows the exposure level of GM37 in csf based on dose andinjection site at the end of the study. FIG. 20D compares the number ofcells with phosphorylated alpha-synuclein positive inclusions countedfrom every sixth section in substantia nigra after treatment with GM37or PBS control. The mice treated with GM37 both 15 mg/kg iv and 30 mg/kgip had a significant reduction in cells with phosphorylatedalpha-synuclein inclusions compared to the PBS treated mice (Example10).

FIG. 21 shows alignment of human α (SEQ ID NO:10), β (SEQ ID NO:37) andγ (SEQ ID NO:38) synuclein proteins. Amino acid residues different fromα-synuclein are highlighted. Gaps are indicated by a dot. SwissProtnumbers are in parenthesis.

FIG. 22 shows alignment of alpha-synuclein orthologs (Cynomolgus monkey,SEQ ID NO:39; Rat, SEQ ID NO:40; Mouse, SEQ ID NO:41). Amino acidresidues different from human alpha-synuclein (SEQ ID NO:10) arehighlighted. SwissProt numbers are shown in parenthesis.

FIG. 23 shows transient expression of GM37 (named GM37 wild type (wt)and 3 GM37 variants, named GM37 var 1, 2 and 3. Asterisk indicates thatthe data are determined post protein A purification and neutralisation.† indicates that data are calculated from yield achieved post protein Aand neutralization in relation to scale of expression culture (0.4 L).

FIG. 24 shows a competition ELISA measuring binding of four antibodiesGM37 wt, GM37 var 1, GM37 var 2 and GM37 var 3 to human alpha-synuclein.Plates coated with alpha-synuclein are used to detect the amount ofantibody remaining after preincubation in solution of each antibody (0.3μg/ml) with increasing concentration of alpha-synuclein (0-1000 nM). Allfour antibodies show similar binding to alpha-synuclein.

FIG. 25 shows a table comparing the binding rate kinetic parameters ofGM37 wt and variants 1-3 to immobilized recombinant humanalpha-synuclein. The binding was measured using SPR and the rates weredetermined using a 1:1 binding algorithm (BIAcore® T200).

FIG. 26 compares the effect of alpha-synuclein antibodies onphosphorylated alpha-synuclein levels in murine primary neurons treatedwith pathological alpha-synuclein fibrillary seeds. Primary neurons weretreated with seeds (1 Ong) in the presence or absence of four GM37, GM37var 1, GM37 var 2 and GM37 var 3 (2 μg). Neurons were fixed & stainedafter 3 weeks and analysed by Cellomics ARRAYSCAN™ for alpha-synucleinphospho serine 129 positive spots. Cells treated with seeds alone orwith seeds plus the isotype control antibody (B12) show significantlyincreased levels phosphorylation. Cells treated with GM37 wt and the 3variants are able to inhibit phosphorylation of alpha-synuclein, theyall show the same level of phosphorylation as cells that did not receiveseeds. Data is shown as mean±SD as determined from seven images per wellin five wells. N=2.

FIG. 27 compares temperature dependent aggregation of wt GM37, var1,var2 and var3. A sample of each of the antibodies was subjected to asteady increase in temperature over time and the level of aggregationwas simultaneously measured by multi-angle light scattering (PrometheusNT.48, NanoTemper Technologies). The temperature for onset ofaggregation is similar for GM37 and GM37-variants, however the lowestlevel of aggregation observed for GM37-Var2.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “alpha-synuclein” is synonymous with “thealpha-synuclein protein” and refers to any of the alpha-synucleinprotein isoforms (identified in, for example, UniProt as P37840, 1-3).The amino acid numbering of alpha-synuclein is given with respect to SEQID NO:10 as shown below, with methionine (M) being amino acid residue1:

SEQ ID NO: 10: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYVGSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQKTVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

The present invention relates to antibodies and to fragments ofantibodies that are capable of specifically binding to alpha-synuclein,and in particular to human alpha-synuclein. In particular, theantibodies and fragments thereof exhibit the ability to specificallybind to an epitope within 112-117 of human alpha-synuclein.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule or according to some embodiments of theinvention, a fragment of an immunoglobulin molecule which has theability to specifically bind to an epitope of a molecule (“antigen”).Naturally occurring antibodies typically comprise a tetramer which isusually composed of at least two heavy (H) chains and at least two light(L) chains. Each heavy chain is comprised of a heavy chain variabledomain (abbreviated herein as VH) and a heavy chain constant domain,usually comprised of three domains (CH1, CH2 and CH3). Heavy chains canbe of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes),IgA (IgA1 and IgA2 subtypes), IgM and IgE. Each light chain is comprisedof a light chain variable domain (abbreviated herein as VL) and a lightchain constant domain (CL). Light chains include kappa chains and lambdachains. The heavy and light chain variable domain is typicallyresponsible for antigen recognition, while the heavy and light chainconstant domain may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions,” that are interspersed with regions of more conserved sequence,termed “framework regions” (FR). Each VH and VL is composed of three CDRDomains and four FR Domains arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.The variable domains of the heavy and light chains contain a bindingdomain that interacts with an antigen. Of particular relevance areantibodies and their antigen-binding fragments that have been “isolated”so as to exist in a physical milieu distinct from that in which it mayoccur in nature or that have been modified so as to differ from anaturally occurring antibody in amino acid sequence.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and linear epitopes aredistinguished in that the binding to the former, but not the latter, isalways lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen-binding peptide (in other words, the amino acidresidue is within the footprint of the specifically antigen-bindingpeptide). The term “112-117 epitope” refers to a region of humanalpha-synuclein that contains at least 4 of the 6 amino acid residues of112-117 human alpha-synuclein, which epitope does not include anyresidue from 1-111 (including any residue from 106-111) of humanalpha-synuclein, nor any residue from 118-140 (including residue118-120) of human alpha-synuclein. As used herein, an antibody is saidto be capable of specifically binding to an epitope within the “112-117epitope” if it is capable of specifically binding to humanalpha-synuclein by binding to at least 4 of the 6 amino acid residues ofthe 112-117 epitope.

As used herein, the term “antigen-binding fragment of an antibody” meansa fragment, portion, region or domain of an antibody (regardless of howit is produced (e.g., via cleavage, recombinantly, synthetically, etc.))that is capable of specifically binding to an epitope, and thus the term“antigen-binding” is intended to mean the same as “epitope-binding” sothat, for example, an “antigen-binding fragment of an antibody” isintended to be the same as an “epitope-binding fragment of an antibody”.An antigen-binding fragment may contain 1, 2, 3, 4, 5 or all 6 of theCDR Domains of such antibody and, although capable of specificallybinding to such epitope, may exhibit a specificity, affinity orselectivity toward such epitope that differs from that of such antibody.Preferably, however, an antigen-binding fragment will contain all 6 ofthe CDR Domains of such antibody. An antigen-binding fragment of anantibody may be part of, or comprise, a single polypeptide chain (e.g.,an scFv), or may be part of, or comprise, two or more polypeptidechains, each having an amino-terminus and a carboxyl terminus (e.g., adiabody, a Fab fragment, a Fab₂ fragment, etc.). Fragments of antibodiesthat exhibit antigen-binding ability can be obtained, for example, byprotease cleavage of intact antibodies. More preferably, although thetwo domains of the Fv fragment, VL and VH, are naturally encoded byseparate genes, or polynucleotides that encode such gene sequences(e.g., their encoding cDNA) can be joined, using recombinant methods, bya flexible linker that enables them to be made as a single protein chainin which the VL and VH regions associate to form monovalentantigen-binding molecules (known as single-chain Fv (scFv); see e.g.,Bird et al; (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. (U.S.A.) 85:5879-5883). Alternatively, by employing aflexible linker that is too short (e.g., less than about 9 residues) toenable the VL and VH regions of a single polypeptide chain to associatetogether, one can form a bispecific antibody, diabody, or similarmolecule (in which two such polypeptide chains associate together toform a bivalent antigen-binding molecule) (see for instance PNAS USA90(14), 6444-8 (1993) for a description of diabodies). Examples ofantigen-binding fragments encompassed within the present inventioninclude (i) a Fab′ or Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains, or a monovalent antibody as described inWO2007059782; (ii) F(ab′)2 fragments, bivalent fragments comprising twoFab fragments linked by a disulfide bridge at the hinge domain; (iii) anFd fragment consisting essentially of the VH and CH1 domains; (iv) a Fvfragment consisting essentially of a VL and VH domains, (v) a dAbfragment (Ward et al., Nature 341, 544-546 (1989)), which consistsessentially of a VH domain and also called domain antibodies (Holt etal; Trends Biotechnol. 2003 November; 2i(II):484-90); (vi) camelid ornanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5_(I):III-24) and (vii) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they may be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain antibodies or single chain Fv (scFv),see for instance Bird et al., Science 242, 423-426 (1988) and Huston etal., PNAS USA 85, 5879-5883 (1988)). These and other useful antibodyfragments in the context of the present invention are discussed furtherherein. It also should be understood that the term antibody, unlessspecified otherwise, also includes antibody-like polypeptides, such aschimeric antibodies and humanized antibodies, and antibody fragmentsretaining the ability to specifically bind to the antigen(antigen-binding fragments) provided by any known technique, such asenzymatic cleavage, peptide synthesis, and recombinant techniques. Anantibody as generated can possess any isotype. As used herein, “isotype”refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3 orIgG4) that is encoded by heavy chain constant domain genes. Suchantibody fragments are obtained using conventional techniques known tothose of skill in the art; suitable fragments capable of binding to adesired epitope may be readily screened for utility in the same manneras an intact antibody.

The term “bispecific antibody” refers to an antibody containing twoindependent antigen-binding fragments that each target independenttargets. These targets can be epitopes present on different proteins ordifferent epitopes present on the same target. Bispecific antibodymolecules can be made using compensatory amino acid changes in theconstant domains of the HCs of the parent monospecific bivalent antibodymolecules. The resulting heterodimeric antibody contains one Fabscontributed from two different parent monospecific antibodies. Aminoacid changes in the Fc domain leads to increased stability of theheterodimeric antibody with bispecificity that is stable over time.(Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran etal., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011),Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineering25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145-5150 (2013),Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecificantibodies can also include molecules that are generated using ScFvfusions. Two monospecific scfv are then independently joined to Fcdomains able to form stable heterodimers to generate a single bispecificmolecule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific moleculeshave dual binding capabilities. For example, targeting both atherapeutic target and a transcytosing surface receptor for the purposeof delivering a therapeutic antibody across the blood brain barrier totreat a CNS disease.

The terms GM37, GM-37, GM37 wild type (wt), mab37 and 6004-37 are usedinterchangeably herein and all refer to the same antibody.

The term antibody GM37 is intended to include an antibody orantigen-binding fragment thereof comprising or consisting of the HeavyChain as given in CDR1-3 SEQ ID Nos:1-3 and the Light Chain CDR1-3 asgiven in SEQ ID Nos:4-6. In one embodiment, the antibody GM37 orantigen-binding fragment thereof may comprise or consist of the heavychain variable domain of SEQ ID NO:7 and/or the light chain variabledomain of SEQ ID NO:8. For example, the antibody GM37 may be an IgGantibody comprising a heavy chain consisting of a variable domain of SEQID NO:7 and a constant domain of SEQ ID NO:18 together with a lightchain consisting of a variable domain of SEQ ID NO:8 and a kappaconstant domain of SEQ ID NO:17.

Deamination of proteins, and in these instance antibodies, can occurspontaneously during manufacturing and storage, but also in vivo, andmakes the quality of the final pharmaceutical medicament difficult tocontrol. The deamination may also in some instances affect the activityof the molecule. Deamination occurs at asparagine residues, but thelocation of the relevant asparagine may be difficult to predict withcertainty, but may be influenced in some instances by anasparagine-glycine motif. Several possible deamination motifs are foundon the GM37 antibody, however, one likely site of deamination was foundto be at residue 54 of the heavy chain. The subsequent substitution ofasparagine by another amino acid is not straight forward, but 3 variantsof GM37 (GM37 variant (var) 1, 2 and 3) were found to retain theactivity of the original GM37 (GM37 wild type (wt)).

The term GM37 variants refers to the deaminated variants 1, 2 or 3,wherein variant 1 has a N54S substitution, variant 2 has a N54Qsubstitution and variant 3 has a N54H compared to the GM37 antibodydescribed herein above.

The antibody GM37 variant (var) 1, 2 and 3 are thus intended to includean antibody or antigen-binding fragment thereof comprising or consistingof the Heavy Chain as given in CDR1 and 3 SEQ ID Nos:1 and 3 from GM 37and the Light Chain CDR1-3 from GM37 as given in SEQ ID Nos:4-6, butdiffering in their heavy chain CDR2 so that variant 1 has CDR 2 of SEQID NO:33, variant 2 has CDR 2 of SEQ ID NO:34 and variant 3 has CDR 2 ofSEQ ID NO:35.

In one embodiment, the antibody GM37 variants or their antigen-bindingfragments may comprise or consist of the heavy chain variable domain ofSEQ ID NO:30, 31 and 32 for variant 1, 2 and 3, respectively, and thelight chain variable domain of SEQ ID NO:8. The antibody GM37 may be anIgG antibody comprising a heavy chain consisting of a variable domain ofSEQ ID NO:30, 31 or 32 and a constant domain of SEQ ID NO:18 togetherwith a light chain consisting of a variable domain of SEQ ID NO:8 and akappa constant domain of SEQ ID NO:17.

The terms GM285, GM-285, mab285 and 6004-285 are used interchangeablyherein and all refer to the same antibody.

The term antibody GM285 is intended to include an antibody orantigen-binding fragment thereof comprising or consisting of the HeavyChain as given in CDR1-3 SEQ ID NOs:20-22 and the Light Chain CDR1-3 asgiven in SEQ ID NOs:23-25. In one embodiment, the antibody GM37 orantigen-binding fragment thereof may comprise or consist of the heavychain variable domain of SEQ ID NO:26 and/or the light chain variabledomain of SEQ ID NO:27. For example, the antibody GM37 may be an IgGantibody comprising a heavy chain consisting of a variable domain of SEQID NO:26 and a constant domain of SEQ ID NO:28 together with a lightchain consisting of a variable domain of SEQ ID NO:27 and a kappaconstant domain of SEQ ID NO:29.

The GM285 antibody specifically binds an epitope within the sequence112-115 (ILED; SEQ ID NO:19) of human alpha-synuclein (SEQ ID NO:10).

Unless otherwise specified herein, the numbering of amino acid residuesin this region is according to IMGT®, the international ImMunoGeneTicsinformation System® or, Kabat, E. A., Wu, T. T., Perry, H. M.,Gottesmann, K. S. & Foeller, C. (1991). Sequences of Proteins ofImmunological Interest, 5th edit., NIH Publication no. 91-3242 U.S.Department of Health and HumanServices. Chothia, C. & Lesk, A. M.(1987). Canonical structures for the hypervariable domains ofimmunoglobulins. J. Mol. Biol. 196, 901-917).

An “anti-alpha-synuclein antibody” or “alpha-synuclein antibody” (usedinterchangeably herein, depending on the context wherein its written) isan antibody or an antigen-binding fragment thereof which bindsspecifically to alpha-synuclein or an alpha-synuclein fragment asdefined herein above, in particular the sequence of alpha-synucleincorresponding to SEQ ID NOs 9 and/or 19.

The term “human antibody” (which may be abbreviated to “humAb” or“HuMab”), as used herein, is intended to include antibodies havingvariable and constant domains derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orduring gene rearrangement or by somatic mutation in vivo).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A conventional monoclonal antibody compositiondisplays a single binding specificity and affinity for a particularepitope. In certain embodiments a monoclonal antibody can be composed ofmore than one Fab domain thereby increasing the specificity to more thanone target. The terms “monoclonal antibody” or “monoclonal antibodycomposition” are not intended to be limited by any particular method ofproduction (e.g., recombinant, transgenic, hybridoma, etc.).

The term “humanized” refer to a molecule, generally prepared usingrecombinant techniques, having an antigen-binding site derived from animmunoglobulin from a non-human species and a remaining immunoglobulinstructure based upon the structure and/or sequence of a humanimmunoglobulin. The antigen-binding site may comprise either completenon-human antibody variable domains fused to human constant domains, oronly the complementarity determining regions (CDRs) of such variabledomains grafted to appropriate human framework regions of human variabledomains. The framework residues of such humanized molecules may be wildtype (e.g., fully human) or they may be modified to contain one or moreamino acid substitutions not found in the human antibody whose sequencehas served as the basis for humanization. Humanization lessens oreliminates the likelihood that a constant domain of the molecule willact as an immunogen in human individuals, but the possibility of animmune response to the foreign variable domain remains (LoBuglio, A. F.et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: KineticsAnd Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).Another approach focuses not only on providing human-derived constantdomains, but modifying the variable domains as well so as to reshapethem as closely as possible to human form. It is known that the variabledomains of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theantigens in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular antigen,the variable domains can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K. et al. (1993) Cancer Res 53:851-856.Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,”Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping HumanAntibodies: Grafting An Antilysozyme Activity,” Science 239:1534-1536;Kettleborough, C. A. et al. (1991) “Humanization Of A Mouse MonoclonalAntibody By CDR-Grafting: The Importance Of Framework Residues On LoopConformation,” Protein Engineering 4:773-3783; Maeda, H. et al. (1991)“Construction Of Reshaped Human Antibodies With HIV-NeutralizingActivity,” Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al.(1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci.(U.S.A.) 88:4181-4185; Tempest, P. R. et al. (1991) “Reshaping A HumanMonoclonal Antibody To Inhibit Human Respiratory Syncytial VirusInfection in vivo,” Bio/Technology 9:266-271; Co, M. S. et al. (1991)“Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci.(U.S.A.) 88:2869-2873; Carter, P. et al. (1992) “Humanization Of AnAnti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci.(U.S.A.) 89:4285-4289; and Co, M. S. et al. (1992) “Chimeric AndHumanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol.148:1149-1154. In some embodiments, humanized antibodies preserve allCDR sequences (for example, a humanized mouse antibody which containsall six CDRs from the mouse antibodies). In other embodiments, humanizedantibodies have one or more CDRs (one, two, three, four, five, six)which are altered with respect to the original antibody, which are alsotermed one or more CDRs “derived from” one or more CDRs from theoriginal antibody. The ability to humanize an antigen is well known(see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205;6,407,213; 6,881,557).

As used herein, an antibody or an antigen-binding fragment thereof issaid to “specifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity or avidity with thatepitope relative to alternative epitopes. In one embodiment, theantibody, or antigen-binding fragment thereof, of the invention binds atleast 10-fold more strongly to its target (human alpha synuclein) thanto another molecule; preferably at least 50-fold more strongly and morepreferably at least 100-fold more strongly. Preferably, the antibody, orantigen-binding fragment thereof, binds under physiological conditions,for example, in vivo. Thus, an antibody that is capable of “specificallybinding” to an epitope within residues 112-117 (ILEDMP (SEQ ID NO:9)) ofhuman alpha-synuclein encompasses an antibody or antigen-bindingfragments thereof, that is capable of binding to an epitope withinresidues 112-117 of human alpha-synuclein with such specificity and/orunder such conditions. Methods suitable for determining such bindingwill be known to those skilled in the art, and exemplary methods aredescribed in the accompanying Examples. As used herein, the term“binding” in the context of the binding of an antibody to apredetermined antigen typically refers to binding with an affinitycorresponding to a KD of about 10⁻⁷ M or less, such as about 10⁻⁸ M orless, such as about 10⁻⁹ M or less when determined by for instancesurface plasmon resonance (SPR) technology in either a BIAcore® 3000 orT200instrument using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a KD that is at least ten-fold lower, such as at least100 fold lower, for instance at least 1,000 fold lower, such as at least10,000 fold lower, for instance at least 100,000 fold lower than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen. The amountwith which the affinity is lower is dependent on the KD of the antibody,so that when the KD of the antibody is very low (that is, the antibodyis highly specific), then the amount with which the affinity for theantigen is lower than the affinity for a non-specific antigen may be atleast 10,000 fold.

The term “kd” (sec −1 or 1/s), as used herein, refers to thedissociation rate constant of a particular antibody-antigen interaction.Said value is also referred to as the koff value.

The term “ka” (M-1×sec-1 or 1/Msec), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.

The term “KD” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the kd by the ka.

The term “KA” (M-1 or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the ka by the kd.

In one embodiment, the invention relates to an antibody orantigen-binding fragments thereof, which exhibits one or more of thefollowing properties:

-   -   i. a binding affinity (KD) for alpha-synuclein of between 0.5-10        nM, such as 1-5 nM or 1-2 nM;    -   ii. capability of inhibiting protease truncation of        alpha-synuclein fibrils;    -   iii. capability of reversing impairment in basal synaptic        transmission in F28-snca transgenic mice;    -   iv. capability of reducing levels of alpha-synuclein in the        mouse hippocampus as measured by in vivo microdialysis;    -   v. capability, when administered chronically, to restore motor        function in a rat model of Parkinson's disease    -   vi. Capability to prevent seeding of alpha-synuclein (such as        accumulation of insoluble phosphorylated alpha-synuclein in        vitro and/or in a mouse model of Parkinson's disease); and/or    -   vii. Capability to bind truncated alpha-synuclein in a human        brain.

The binding affinity (KD) for alpha-synuclein may be determined usingmethods well known in the art, e.g. as described in Example 2.

The term “capability of inhibiting protease truncation ofalpha-synuclein fibrils” includes the capability of inhibiting calpain-1induced formation of fragment 1-119-122 of human alpha synuclein inprimary cortical neurons (see Example 5).

The term “capability of reversing impairment in basal synaptictransmission in F28-snca transgenic mice” includes the capability ofreverse the impairment of synaptic transmission and plasticity in theCA1 area of the hippocampus in F28-snca transgenic mice, for example asindicated by evoked fEPSP slope as measured electrophysiologically (SeeExample 6).

The term “capability of reducing levels of alpha-synuclein in the mousehippocampus as measured by in vivo microdialysis” includes thecapability of reducing levels of human alpha synuclein in thehippocampus awake, freely-moving F28-snca transgenic mice, as measuredusing in vivo microdialysis (see Example 7).

The term “capability, when administered chronically, to restore motorfunction in a rat model of Parkinson's disease” include the capabilityto reduce or eliminate motor asymmetry in a rat recombinantadeno-associated viral vector (rAAV) model of Parkinson's Disease (seeExample 8).

In some antibodies, only part of a CDR, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting therelevant epitope and not in the SDRs can be identified based on previousstudies (for example residues H60-H65 in CDR H2 are often not required),from regions of Kabat CDRs lying outside Chothia hypervariable loops(see, Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST, National Institutes of Health Publication No. 91-3242;Chothia, C. et al. (1987) “Canonical Structures For The Hypervariabledomains Of Immunoglobulins,” J. Mol. Biol. 196:901-917), by molecularmodeling and/or empirically, or as described in Gonzales, N. R. et al.(2004) “SDR Grafting Of A Murine Antibody Using Multiple Human GermlineTemplates To Minimize Its Immunogenicity,” Mol. Immunol. 41:863-872. Insuch humanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The fact that a single amino acid alteration of a CDR residue can resultin loss of functional binding (Rudikoff, S. etc. (1982) “Single AminoAcid Substitution Altering Antigen-Binding Specificity,” Proc. Natl.Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematicallyidentifying alternative functional CDR sequences. In one preferredmethod for obtaining such variant CDRs, a polynucleotide encoding theCDR is mutagenized (for example via random mutagenesis or by asite-directed method (e.g., polymerase chain-mediated amplification withprimers that encode the mutated locus)) to produce a CDR having asubstituted amino acid residue. By comparing the identity of therelevant residue in the original (functional) CDR sequence to theidentity of the substituted (non-functional) variant CDR sequence, theBLOSUM62.iij substitution score for that substitution can be identified.The BLOSUM system provides a matrix of amino acid substitutions createdby analyzing a database of sequences for trusted alignments (Eddy, S. R.(2004) “Where Did The BLOSUM62 Alignment Score Matrix Come From?,”Nature Biotech. 22(8):1035-1036; Henikoff, J. G. (1992) “Amino acidsubstitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA)89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,S. F. (1991) “Amino Acid Substitution Matrices From An InformationTheoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the mostadvanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table1 presents the BLOSUM62.iij substitution scores (the higher the scorethe more conservative the substitution and thus the more likely thesubstitution will not affect function). If an antigen-binding fragmentcomprising the resultant CDR fails to bind to alpha-synuclein, forexample, then the BLOSUM62.iij substitution score is deemed to beinsufficiently conservative, and a new candidate substitution isselected and produced having a higher substitution score. Thus, forexample, if the original residue was glutamate (E), and thenon-functional substitute residue was histidine (H), then theBLOSUM62.iij substitution score will be 0, and more conservative changes(such as to aspartate, asparagine, glutamine, or lysine) are preferred.

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A +4 −1 −2 −2 0 −1 −1 0−2 −1 −1 −1 −1 −2 −1 +1 0 −3 −2 0 R −1 +5 0 −2 −3 +1 0 −2 0 −3 −2 +2 −1−3 −2 −1 −1 −3 −2 −3 N −2 0 +6 +1 −3 0 0 0 +1 −3 −3 0 −2 −3 −2 +1 0 −4−2 −3 D −2 −2 +1 +6 −3 0 +2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3−3 −3 +9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 +1 0 0 −3 +5+2 −2 0 −3 −2 +1 0 −3 −1 0 −1 −2 −1 −2 E −1 0 0 +2 −4 +2 +5 −2 0 −3 −3+1 −2 −3 −1 0 −1 −3 −2 −2 G 0 −2 0 −1 −3 −2 −2 +6 −2 −4 −4 −2 −3 −3 −2 0−2 −2 −3 −3 H −2 0 +1 −1 −3 0 0 −2 +8 −3 −3 −1 −2 −1 −2 −1 −2 −2 +2 −3 I−1 −3 −3 −3 −1 −3 −3 −4 −3 +4 +2 −3 +1 0 −3 −2 −1 −3 −1 +3 L −1 −2 −3 −4−1 −2 −3 −4 −3 +2 +4 −2 +2 0 −3 −2 −1 −2 −1 +1 K −1 +2 0 −1 −3 +1 +1 −2−1 −3 −2 +5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2 −3 −1 0 −2 −3 −2 +1 +2 −1+5 0 −2 −1 −1 −1 −1 +1 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 +6 −4 −2 −2+1 +3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 +7 −1 −1 −4 −3 −2 S+1 −1 +1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 +4 +1 −3 −2 −2 T 0 −1 0 −1 −1 −1−1 −2 −2 −1 −1 −1 −1 −2 −1 +1 +5 −2 −2 0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3−2 −3 −1 +1 −4 −3 −2 +11 +2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3 +2 −1 −1 −2 −1+3 −3 −2 −2 +2 +7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 +3 +1 −2 +1 −1 −2 −2 0−3 −1 +4

The invention thus contemplates the use of random mutagenesis toidentify improved CDRs. In the context of the present invention,conservative substitutions may be defined by substitutions within theclasses of amino acids reflected in one or more of the following threetables:

Amino Acid Residue Classes for Conservative Substitutions:

TABLE 2 Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg(R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn(N), and Gln (Q) Aliphatic Uncharged Residues Cly (G), Ala (A), Val (V),Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), andPro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)Alternative Conservative Amino Acid Residue Substitution Classes:

TABLE 3 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y WAlternative Physical and Functional Classifications of Amino AcidResidues:

TABLE 4 Alcohol Group-Containing Residues S and T Aliphatic Residues I,L, V and M Cycloalkenyl-Associated Residues F, H, W and Y HydrophobicResidues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively ChargedResidues D and E Polar Residues C, D, E, H, K, N, Q, R, S and TPositively Charged Residues H, K and R Small Residues A, C, D, G, N, P,S, T and V Very Small Residues A, G and S Residues Involved In TurnFormation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible Residues Q,T, K, S, G, P, D, E and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2 d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (ordecrease) CDR affinity. This technology, referred to as affinitymaturation, employs mutagenesis or “CDR walking” and re-selection usesthe target antigen or an antigenic antigen-binding fragment thereof toidentify antibodies having CDRs that bind with higher (or lower)affinity to the antigen when compared with the initial or parentalantibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased (or decreased) binding affinity for theantigen can be screened by contacting the immobilized mutants withlabeled antigen. Any screening method known in the art can be used toidentify mutant antibodies with increased or decreased affinity to theantigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci.(U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDRwalking which randomizes the Light Chain may be used possible (see,Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described forexample in: Krause, J. C. et al. (2011) “An Insertion Mutation ThatDistorts Antibody Binding Site Architecture Enhances Function Of A HumanAntibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan,C. T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB RecombinantImmunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDRComposition Biases Enrich Binder Functionality Landscapes,” J. Mol.Biol. 401(1):84-96; Montgomery, D. L. et al. (2009) “Affinity MaturationAnd Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,”MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation ByTargeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab DerivedFrom A Synthetic Naïve Human Antibody Library And Directed Against TheInternal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With ImprovedHIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119;Finlay, W. J. et al. (2009) “Affinity Maturation Of A Humanized RatAntibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A HighLevel Of Mutational Plasticity Both Inside And Outside TheComplementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558;Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity AndSpecificity For Therapeutic Development,” Methods Mol. Biol.525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation OfHuman GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol.46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation OfAntibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA)105(26):9029-9034.

Thus, the sequence of CDR variants of encompassed antibodies or theirantigen-binding fragments may differ from the sequence of the CDR of theparent antibody, GM37, GM37 var 1-3, or 285, through substitutions; forinstance substituted 4 amino acid residue, 3 amino acid residue, 2 aminoacid residue or 1 of the amino acid residues. According to an embodimentof the invention it is furthermore envisaged that the amino acids in theCDR regions may be substituted with conservative substitutions, asdefined in the 3 tables above.

The term “treatment” or “treating” as used herein means ameliorating,slowing, attenuating or reversing the progress or severity of a diseaseor disorder, or ameliorating, slowing, attenuating or reversing one ormore symptoms or side effects of such disease or disorder. For purposesof this invention, “treatment” or “treating” further means an approachfor obtaining beneficial or desired clinical results, where “beneficialor desired clinical results” include, without limitation, alleviation ofa symptom, diminishment of the extent of a disorder or disease,stabilized (i.e., not worsening) disease or disorder state, delay orslowing of the progression a disease or disorder state, amelioration orpalliation of a disease or disorder state, and remission of a disease ordisorder, whether partial or total, detectable or undetectable.

An “effective amount,” when applied to an antibody or antigen-bindingfragments thereof, of the invention, refers to an amount sufficient, atdosages and for periods of time necessary, to achieve an intendedbiological effect or a desired therapeutic result including, withoutlimitation, clinical results. The phrase “therapeutically effectiveamount” when applied to an antibody or antigen-binding fragmentsthereof, of the invention is intended to denote an amount of theantibody, or antigen-binding fragment thereof, that is sufficient toameliorate, palliate, stabilize, reverse, slow, attenuate or delay theprogression of a disorder or disease state, or of a symptom of thedisorder or disease. In an embodiment, the method of the presentinvention provides for administration of the antibody, orantigen-binding fragment thereof, in combinations with other compounds.In such instances, the “effective amount” is the amount of thecombination sufficient to cause the intended biological effect.

A therapeutically effective amount of an anti-alpha-synuclein antibodyor antigen-binding fragment thereof of the invention may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the anti-alpha-synuclein antibody toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody or antibody portion are outweighed by the therapeuticallybeneficial effects.

As indicated above, the present invention particularly relates to amonoclonal antibody capable of specifically binding to an epitope withinamino acids 112-117 of human alpha-synuclein (SEQ ID NO:9 (ILEDMP)). Inone embodiment the antibody is capable of competing with the antibodyGM37 for binding to an epitope within the 112-117 amino acids ofalpha-synuclein.

The antibodies of the present invention, exemplified by GM37 itsvariants GM37 var 1-3 and GM285, and their alpha-synuclein bindingfragments are capable of binding the toxic alpha-synuclein fragmentconsisting of residues 1-119/122 of alpha-synuclein and neutralizing itstoxicity (for example, by extracellular binding to the alpha-synucleinfragment and thereby preventing it from being taken up by cells.Surprisingly the antibodies of the present invention, which are capableof binding to an epitope within amino acids 112-117 of alpha-synucleinare superior to prior art antibodies such as antibody 9E4 in binding totoxic alpha-synuclein species in human brain, and have superior effectson clearing extracellular alpha-synuclein and normalising an impairedsynaptic transmission induced by alpha-synuclein in vivo. The antibodiesof the invention are also able to ameliorate the appearance of arelevant motor phenotype in a rat model for Parkinson's disease.

The antibodies of the present invention are preferably human orhumanized antibodies.

The present invention also provides a method of reducing alpha-synucleinaggregate formation in a patient, comprising administering to thepatient in need of such treatment, a therapeutically effective amount ofan antibody of the invention.

Further the antibodies may be in a composition together with apharmaceutically acceptable carrier, diluent and/or stabilizer. Theantibodies of the invention may be used in therapy. In particular, theantibodies of the invention may be used in treating synucleinopathiessuch as Parkinson's disease (including idiopathic inherited forms ofParkinson's disease), Gaucher's Disease, Diffuse Lewy Body Disease(DLBD), Lewy body variant of Alzheimer's disease (LBV), CombinedAlzheimer's and Parkinson disease, pure autonomic failure and multiplesystem atrophy.

The treatment envisioned by the present invention may be chronic and thepatient may be treated at least 2 weeks, such as at least for 1 month,6, months, 1 year or more.

The antibodies of antigen-binding fragments thereof of the presentinvention may be produced in different cell lines, such as a human cellline, a mammal non-human cell line, and insect cell line, for example aCHO cell line, HEK cell line, BHK-21 cell line, murine cell line (suchas a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line,HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al,2015, Crit Rev Biotechnol. September 18:1-13., the contents which isincluded herein by reference).

The antibodies of the present invention may for example be monoclonalantibodies produced by the hybridoma method first described by Kohler etal., Nature 256, 495 (1975), or may be produced by recombinant DNAmethods. Monoclonal antibodies may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222,581-597 (1991). Monoclonal antibodies may be obtained from any suitablesource. Thus, for example, monoclonal antibodies may be obtained fromhybridomas prepared from murine splenic B lymphocyte cells obtained frommice immunized with an antigen of interest, for instance, in the form ofcells expressing the antigen on the surface, or a nucleic acid encodingan antigen of interest. Monoclonal antibodies may also be obtained fromhybridomas derived from antibody-expressing cells of immunized humans ornon-human mammals such as rats, rabbits, dogs, sheep, goats, primates,etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against alpha-synuclein may begenerated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. Such transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively.

The HuMAb mouse contains a human immunoglobulin gene minilocus thatencodes unrearranged human heavy variable and constant (μ and Y) andlight variable and constant (κ) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous μ and κ chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or Igκ and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825,5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318,5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227,WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al., EMBOJ. 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424), and a KCo5 human kappalight chain transgene (as described in Fishwild et al., NatureBiotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice have aHCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424), the HCo17 mice have a HCo17human heavy chain transgene (as described in Example 2 of WO 01/09187)and the HCo20 mice have a HCo20 human heavy chain transgene. Theresulting mice express human immunoglobulin heavy and kappa light chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can begenerated by crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) asdescribed in WO 09/097006.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain trans-chromosomecomposed of chromosome 14 antigen-binding fragment hCF (SC20) asdescribed in WO 02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172and 5,741,957.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant domains, kappa or lambda, maybe used. If desired, the class of an anti-alpha-synuclein antibody ofthe present invention may be switched by known methods. For example, anantibody of the present invention that was originally IgM may be classswitched to an IgG antibody of the present invention. Further, classswitching techniques may be used to convert one IgG subclass to another,for instance from IgGI to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3 or IgG4 antibody for various therapeuticuses. In one embodiment an antibody of the present invention is an IgG1antibody, for instance an IgG1, κ. An antibody is said to be of aparticular isotype if its amino acid sequence is most homologous to thatisotype, relative to other isotypes.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG antibody, in particular an IgG1, κ antibody.In another embodiment, the antibody of the invention is an antibodyfragment or a single-chain antibody.

Antibodies and antigen-binding fragments thereof may e.g. be obtained byantigen-binding fragmentation using conventional techniques, andantigen-binding fragments screened for utility in the same manner asdescribed herein for whole antibodies. For example, F(ab′)2antigen-binding fragments may be generated by treating antibody withpepsin. The resulting F(ab′)2 antigen-binding fragment may be treated toreduce disulfide bridges to produce Fab′ antigen-binding fragments. Fabantigen-binding fragments may be obtained by treating an IgG antibodywith papain; Fab′ antigen-binding fragments may be obtained with pepsindigestion of IgG antibody. An F(ab′) antigen-binding fragment may alsobe produced by binding Fab′-described below via a thioether bond or adisulfide bond. A Fab′ antigen-binding fragment is an antibodyantigen-binding fragment obtained by cutting a disulfide bond of thehinge domain of the F(ab′)2. A Fab′-antigen-binding fragment may beobtained by treating an F(ab′)2 antigen-binding fragment with a reducingagent, such as dithiothreitol. Antibody antigen-binding fragment mayalso be generated by expression of nucleic acids encoding suchantigen-binding fragments in recombinant cells (see for instance Evanset al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimericgene encoding a portion of an F(ab′)2 antigen-binding fragment couldinclude DNA sequences encoding the CH1 region and hinge domain of the Hchain, followed by a translational stop codon to yield such a truncatedantibody antigen-binding fragment molecule.

In one embodiment, the anti-alpha-synuclein antibody is a monovalentantibody, preferably a monovalent antibody as described in WO2007059782(which is incorporated herein by reference in its entirety) having adeletion of the hinge domain. Accordingly, in one embodiment, theantibody is a monovalent antibody, wherein said anti-alpha-synucleinantibody is constructed by a method comprising: i) providing a nucleicacid construct encoding the light chain of said monovalent antibody,said construct comprising a nucleotide sequence encoding the VL regionof a selected antigen specific anti-alpha-synuclein antibody and anucleotide sequence encoding the constant CL region of an Ig, whereinsaid nucleotide sequence encoding the VL region of a selected antigenspecific antibody and said nucleotide sequence encoding the CL region ofan Ig are operably linked together, and wherein, in case of an IgG1subtype, the nucleotide sequence encoding the CL region has beenmodified such that the CL region does not contain any amino acidscapable of forming disulfide bonds or covalent bonds with other peptidescomprising an identical amino acid sequence of the CL region in thepresence of polyclonal human IgG or when administered to an animal orhuman being; ii) providing a nucleic acid construct encoding the heavychain of said monovalent antibody, said construct comprising anucleotide sequence encoding the VH region of a selected antigenspecific antibody and a nucleotide sequence encoding a constant CHregion of a human Ig, wherein the nucleotide sequence encoding the CHregion has been modified such that the region corresponding to the hingedomain and, as required by the Ig subtype, other regions of the CHregion, such as the CH3 region, does not comprise any amino acidresidues which participate in the formation of disulphide bonds orcovalent or stable non-covalent inter-heavy chain bonds with otherpeptides comprising an identical amino acid sequence of the CH region ofthe human Ig in the presence of polyclonal human IgG or whenadministered to an animal human being, wherein said nucleotide sequenceencoding the VH region of a selected antigen specific antibody and saidnucleotide sequence encoding the CH region of said Ig are operablylinked together; iii) providing a cell expression system for producingsaid monovalent antibody; iv) producing said monovalent antibody byco-expressing the nucleic acid constructs of (i) and (ii) in cells ofthe cell expression system of (iii).

Similarly, in one embodiment, the anti-alpha-synuclein antibody is amonovalent antibody, which comprises:

-   (i) a variable domain of an antibody of the invention as described    herein or an antigen-binding part of the said region, and-   (ii) a CH domain of an immunoglobulin or a domain thereof comprising    the CH2 and CH3 domains, wherein the CH domain or domain thereof has    been modified such that the domain corresponding to the hinge domain    and, if the immunoglobulin is not an IgG4 subtype, other domains of    the CH domain, such as the CH3 domain, do not comprise any amino    acid residues, which are capable of forming disulfide bonds with an    identical CH domain or other covalent or stable non-covalent    inter-heavy chain bonds with an identical CH domain in the presence    of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalentanti-alpha-synuclein antibody has been modified such that the entirehinge domain has been deleted.

In another further embodiment, the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

The invention also includes “Bispecific Antibodies,” wherein ananti-Alpha-synuclein binding region (e.g., a Alpha-synuclein-bindingregion of an anti-alpha-synuclein monoclonal antibody) is part of abivalent or polyvalent bispecific scaffold that targets more than oneepitope, (for example a second epitope could comprise an epitope of anactive transport receptor, such that the Bispecific Antibody wouldexhibit improved transcytosis across a biological barrier, such as theBlood Brain Barrier). Thus, in another further embodiment, themonovalent Fab of an anti-synuclein antibody may be joined to anadditional Fab or scfv that targets a different protein to generate abispecific antibody. A bispecific antibody can have a dual function, forexample a therapeutic function imparted by an anti-synuclein bindingdomain and a transport function that can bind to a receptor molecule toenhance transfer cross a biological barrier, such as the blood brainbarrier.

Anti-alpha-synuclein antibodies, and antigen-binding fragments thereof,of the invention also include single chain antibodies. Single chainantibodies are peptides in which the heavy and light chain Fv regionsare connected. In one embodiment, the present invention provides asingle-chain Fv (scFv) wherein the heavy and light chains in the Fv ofan anti-alpha-synuclein antibody of the present invention are joinedwith a flexible peptide linker (typically of about 10, 12, 15 or moreamino acid residues) in a single peptide chain. Methods of producingsuch antibodies are described in for instance U.S. Pat. No. 4,946,778,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994),Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85,5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990). Thesingle chain antibody may be monovalent, if only a single VH and VL areused, bivalent, if two VH and VL are used, or polyvalent, if more thantwo VH and VL are used.

The anti-alpha-synuclein antibodies and antigen-binding fragmentsthereof described herein may be modified by inclusion of any suitablenumber of modified amino acids and/or associations with such conjugatedsubstituents. Suitability in this context is generally determined by theability to at least substantially retain the alpha-synuclein selectivityand/or the anti-alpha-synuclein specificity associated with thenon-derivatized parent anti-alpha-synuclein antibody. The inclusion ofone or more modified amino acids may be advantageous in, for example,increasing polypeptide serum half-life, reducing polypeptideantigenicity, or increasing polypeptide storage stability. Amino acid(s)are modified, for example, co-translationally or post-translationallyduring recombinant production (e.g., N-linked glycosylation at N—X-S/Tmotifs during expression in mammalian cells) or modified by syntheticmeans. Non-limiting examples of a modified amino acid include aglycosylated amino acid, a sulfated amino acid, a prenylated (e. g.,farnesylated, geranylgeranylated) amino acid, an acetylated amino acid,an acylated amino acid, a PEGylated amino acid, a biotinylated aminoacid, a carboxylated amino acid, a phosphorylated amino acid, and thelike. References adequate to guide one of skill in the modification ofamino acids are replete throughout the literature. Example protocols arefound in Walker (1998) Protein Protocols On CD-Rom, Humana Press,Totowa, N.J. The modified amino acid may, for instance, be selected froma glycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, or an amino acid conjugated to an organicderivatizing agent.

Anti-alpha-synuclein antibodies may also be chemically modified bycovalent conjugation to a polymer to for instance increase theircirculating half-life. Exemplary polymers, and methods to attach them topeptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106,4,179,337, 4,495,285 and 4,609,546. Additional illustrative polymersinclude polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., aPEG with a molecular weight of between about 1,000 and about 40,000,such as between about 2,000 and about 20,000, e.g., about 3,000-12,000g/mol).

The antibodies of the present invention may further be used in adiagnostic method or as a diagnostic imaging ligand.

In one embodiment, anti-alpha-synuclein antibodies comprising one ormore radiolabeled amino acids are provided. A radiolabeledanti-alpha-synuclein antibody may be used for both diagnostic andtherapeutic purposes (conjugation to radiolabeled molecules is anotherpossible feature). Non-limiting examples of such labels include, but arenot limited to bismuth (²¹³Bi), carbon (¹¹C, ¹³C, ¹⁴C), chromium (⁵¹Cr),cobalt (⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu), dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er),fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium, (¹⁶⁶Ho) hydrogen (³H), indium(¹¹¹In, ¹¹²In, ¹¹³In, ¹¹⁵In), iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), iridium(¹⁹²Ir), iron (⁵⁹Fe), krypton (^(81m)Kr), lanthanium (¹⁴⁰La) lutetium(¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), nitrogen (¹³N, ¹⁵N),oxygen (¹⁵O), palladium (¹⁰³Pd), phosphorus (³²P), potassium (⁴²K),praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re) rhodium(¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb), ruthenium (⁸²Ru, ⁹⁷Ru), samarium(¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), sodium (²⁴Na), strontium(⁸⁵Sr, ⁸⁹Sr, ⁹²Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Tl),tin (¹¹³Sn, ¹¹⁷Sn), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb),yttrium (⁹⁰Y) and zinc (⁶⁵Zn). Methods for preparing radiolabeled aminoacids and related peptide derivatives are known in the art (see forinstance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686(2nd edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S.Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (U.S. RE35,500),U.S. Pat. Nos. 5,648,471 and 5,697,902. For example, a radioisotope maybe conjugated by a chloramine T method (Lindegren, S. et al. (1998)“Chloramine-T In High-Specific-Activity Radioiodination Of AntibodiesUsing N-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,”Nucl. Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-SpecificConjugation Of A Radioiodinated Phenethylamine Derivative To AMonoclonal Antibody Results In Increased Radioactivity Localization InTumor,” J. Med. Chem. 36(9):1255-1261; Rea, D. W. et al. (1990)“Site-specifically radioiodinated antibody for targeting tumors,” CancerRes. 50(3 Suppl):857s-861s).

The invention also provides anti-alpha-synuclein antibodies andantigen-binding fragments thereof that are detectably labeled using afluorescent label (such as a rare earth chelate (e.g., a europiumchelate)), a fluorescein-type label (e.g., fluorescein, fluoresceinisothiocyanate, 5-carboxyfluorescein, 6-carboxy fluorescein,dichlorotriazinylamine fluorescein), a rhodamine-type label (e.g., ALEXAFLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XLFLUOROCHROME™ (Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; acyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or ananalogue thereof, all of which are suitable for optical detection.Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin.

Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin. Paramagnetic labelscan also be employed, and are preferably detected using PositronEmission Tomography (PET) or Single-Photon Emission Computed Tomography(SPECT). Such paramagnetic labels include, but are not limited tocompounds containing paramagnetic ions of Aluminum (Al), Barium (Ba),Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu),Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesium(Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os),Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru),Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm),Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), andparticularly, Co⁺², CR⁺², Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺²,Ti⁺³, V⁺³, and V⁺⁴, positron emitting metals using various positronemission tomographies, and non-radioactive paramagnetic metal ions.

Thus in one embodiment the anti-alpha-synuclein antibody of theinvention may be labelled with a fluorescent label, a chemiluminescentlabel, a paramagnetic label, a radioisotopic label or an enzyme label.The labelled antibody may be used in detecting or measuring the presenceor amount of said alpha-synuclein in the brain of a subject. This methodmay comprise the detection or measurement of in vivo imaging ofanti-alpha-synuclein antibody bound to said alpha-synuclein and maycomprises ex vivo imaging of said anti-alpha-synuclein antibody bound tosaid alpha-synuclein.

In a further aspect, the invention relates to an expression vectorencoding one or more polypeptide chains of an antibody of the inventionor an antigen-binding fragment thereof. Such expression vectors may beused for recombinant production of the antibodies and antigen-bindingfragments of the invention.

An expression vector in the context of the present invention may be anysuitable DNA or RNA vector, including chromosomal, non-chromosomal, andsynthetic nucleic acid vectors (a nucleic acid sequence comprising asuitable set of expression control elements). Examples of such vectorsinclude derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,yeast plasmids, vectors derived from combinations of plasmids and phageDNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-alpha-synuclein antibody-encoding nucleic acid is comprised in anaked DNA or RNA vector, including, for example, a linear expressionelement (as described in, for instance, Sykes and Johnston, Nat Biotech12, 355-59 (1997)), a compacted nucleic acid vector (as described in forinstance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vectorsuch as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sizednucleic acid vector (as described in, for instance, Schakowski et al.,Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vectorconstruct, such as a CaPO₄-precipitated construct (as described in, forinstance, WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55(1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson,Somatic Cell Genetics 2, 603 (1981)). Such nucleic acid vectors and theusage thereof are well known in the art (see for instance U.S. Pat. Nos.5,589,466 and 5,973,972).

In one embodiment, the vector is suitable for expression ofanti-alpha-synuclein antibodies or antigen-binding fragments thereof ina bacterial cell. Examples of such vectors include expression vectorssuch as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, JBiol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison, Wis.)and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358(2012), Celik, E. et al. Biotechnol. Adv. 30(5), 1108-1118 (2012), Li,P. et al. Appl. Biochem. Biotechnol. 142(2), 105-124 (2007), Boer, E. etal. Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007), van der Vaart,J. M. Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P. MethodsMol. Biol. 178, 349-357 (2002)).

In an expression vector of the invention, anti-alpha-synucleinantibody-encoding nucleic acids may comprise or be associated with anysuitable promoter, enhancer, and other expression-facilitating elements.Examples of such elements include strong expression promoters (e.g.,human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, andHIV LTR promoters), effective poly (A) termination sequences, an originof replication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

The antibodies of antigen-binding fragments thereof of the presentinvention may be produced in different cell lines, such as a human cellline, a mammal non-human cell line, and insect cell line, for example aCHO cell line, HEK cell line, BHK-21 cell line, murine cell line (suchas a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line,HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al,2015, Crit Rev Biotechnol. September 18:1-13., the contents which isincluded herein by reference).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody or an antigen-binding domain thereof of theinvention as defined herein or a bispecific molecule of the invention asdefined herein. Examples of host cells include yeast, bacteria, andmammalian cells, such as CHO or HEK cells. For example, in oneembodiment, the present invention provides a cell comprising a nucleicacid stably integrated into the cellular genome that comprises asequence coding for expression of an anti-alpha-synuclein antibody ofthe present invention or an antigen-binding fragment thereof. In anotherembodiment, the present invention provides a cell comprising anon-integrated nucleic acid, such as a plasmid, cosmid, phagemid, orlinear expression element, which comprises a sequence coding forexpression of an anti-alpha-synuclein antibody of the invention.

In a further aspect, the invention relates to a method for producing ananti-alpha-synuclein antibody of the invention, said method comprisingthe steps of a) culturing a hybridoma or a host cell of the invention asdescribed herein above, and b) purifying the antibody of the inventionfrom the culture media.

In one embodiment, the invention relates to a preparation that, as suchterm is used herein, comprises an anti-alpha-synuclein antibody asdefined herein, and that is substantially free of naturally-arisingantibodies that are either not capable of binding to alpha-synuclein orthat do not materially alter the anti-alpha-synuclein functionality ofthe preparation. Thus, such a preparation does not encompassnaturally-arising serum, or a purified derivative of such serum, thatcomprises a mixture of an anti-alpha-synuclein antibody and anotherantibody that does not alter the functionality of theanti-alpha-synuclein antibody of the preparation; wherein suchfunctionality is selected from the group consisting of:

-   -   (i) a binding affinity (KD) of the anti-alpha-synuclein antibody        for alpha-synuclein;    -   (ii) a capability of the anti-alpha-synuclein antibody of        inhibiting protease truncation of alpha-synuclein fibrils;    -   (iii) a capability of the anti-alpha-synuclein antibody of        reversing impairment in basal synaptic transmission in F28-snca        transgenic mice;    -   (iv) a capability of the anti-alpha-synuclein antibody of        reducing levels of alpha-synuclein in the mouse hippocampus as        measured by in vivo microdialysis; and    -   (v) a capability, when administered chronically, of the        anti-alpha-synuclein antibody to restore motor function in a rat        model of Parkinson's disease.    -   (vi) a capability to prevent seeding of alpha-synuclein (such as        accumulation of insoluble phosphorylated alpha-synuclein in        vitro and/or in a mouse model of Parkinson's disease); and/or    -   (vii) a capability to bind truncated alpha-synuclein in a human        brain.

The invention particularly relates to preparations of such ananti-alpha-synuclein antibody having a structural change in its aminoacid sequence (in any of its CDRs, variable domains, framework residuesand/or constant domains) relative to the structure of anaturally-occurring anti-alpha-synuclein antibody, wherein saidstructural change causes the anti-alpha-synuclein monoclonal antibody toexhibit a markedly altered functionality (i.e., more than a 20%difference, more than a 40% difference, more than a 60% difference, morethan an 80% difference, more than a 100% difference, more than a 150%difference, more than a 2-fold difference, more than a 4-folddifference, more than a 5-fold difference, or more than a 10-folddifference in functionality) relative to the functionality exhibited bysaid naturally-occurring anti-alpha-synuclein antibody; wherein suchfunctionality is:

-   -   (i) a binding affinity (KD) of the anti-alpha-synuclein        monoclonal antibody for alpha-synuclein;    -   (ii) a capability of the anti-alpha-synuclein monoclonal        antibody of inhibiting protease truncation of alpha-synuclein        fibrils;    -   (iii) a capability of the anti-alpha-synuclein monoclonal        antibody of reversing impairment in basal synaptic transmission        in F28-snca transgenic mice;    -   (iv) a capability of the anti-alpha-synuclein monoclonal        antibody of reducing levels of alpha-synuclein in the mouse        hippocampus as measured by in vivo microdialysis; and/or    -   (v) a capability, when administered chronically, of the        anti-alpha-synuclein monoclonal antibody to restore motor        function in a rat model of Parkinson's disease;    -   (vi) a capability to prevent seeding of alpha-synuclein (such as        accumulation of insoluble phosphorylated alpha-synuclein in        vitro and/or in a mouse model of Parkinson's disease); and/or    -   (vii) a capability to bind truncated alpha-synuclein in a human        brain.        especially wherein such altered functionality is a result of the        structural change and thus is inseparable from it.

The term “substantially free” of naturally-arising antibodies refers tothe complete absence of such naturally-arising antibodies in suchpreparations, or of the inclusion of a concentration of suchnaturally-arising antibodies in such preparations that does notmaterially affect the alpha-synuclein-binding properties of thepreparations. An antibody is said to be “isolated” if it has nonaturally-arising counterpart or has been separated or purified fromcomponents which naturally accompany it.

The term “naturally-arising antibodies,” as it relates to suchpreparations, refers to antibodies (including naturally-arisingautoantibodies) elicited within living humans or other animals, as anatural consequence to the functioning of their immune systems.

Thus, the preparations of the present invention do not exclude, andindeed explicitly encompass, such preparations that contain ananti-alpha-synuclein antibody and a deliberately added additionalantibody capable of binding to an epitope that is not possessed byalpha-synuclein. Such preparations particularly include embodimentsthereof wherein the preparation exhibits enhanced efficacy in treatingsynucleinopathies such as Parkinson's disease (including idiopathic andinherited form of Parkinson's disease), Gaucher's Disease, Diffuse LewyBody Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),Combined Alzheimer's and Parkinson disease, pure autonomic failure andmultiple system atrophy.

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

-   (i) an anti-alpha-synuclein antibody or antigen-binding fragment    thereof, both as defined herein or a preparation, as such term is    defined herein, that comprises such an anti-alpha-synuclein antibody    or antigen-binding fragment thereof, and-   (ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 22ndEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2013.

Pharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on epitope binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a non-ionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition. The diluent is selected to not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers andthe like. The compositions may also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes).

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode, including: parenteral, topical, oral or intranasal means forprophylactic and/or therapeutic treatment. In one embodiment, apharmaceutical composition of the present invention is administeredparenterally. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art. In one embodiment thatpharmaceutical composition is administered by intravenous orsubcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays antibody absorption, for example,monostearate salts and gelatin. Sterile injectable solutions may beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses describedherein are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. Parenteral compositions may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the antialpha-synuclein antibodies depend on the disease or condition to betreated and may be determined by the persons skilled in the art. On anygiven day that a dosage is given, the dosage may range from about 0.0001to about 100 mg/kg, and more usually from about 0.01 to about 5 mg/kg,of the host body weight. For example, dosages can be 1 mg/kg body weightor 10 mg/kg body weight or within the range of 1-10 mg/kg body weight.Exemplary dosages thus include: from about 0.1 to about 10 mg/kg/bodyweight, from about 0.1 to about 5 mg/kg/body weight, from about 0.1 toabout 2 mg/kg/body weight, from about 0.1 to about 1 mg/kg/body weight,for instance about 0.15 mg/kg/body weight, about 0.2 mg/kg/body weight,about 0.5 mg/kg/body weight, about 1 mg/kg/body weight, about 1.5mg/kg/body weight, about 2 mg/kg/body weight, about 5 mg/kg/body weight,or about 10 mg/kg/body weight.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of theanti-alpha-synuclein antibody employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, a suitable daily dose of a compositionof the present invention will be that amount of the compound which isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous. If desired, the effective daily dose of apharmaceutical composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalcomposition as described above.

Labelled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases or disorders. The inventionprovides for the detection or diagnosis of a neurodegenerative orcognitive disease or disorder, including but not limited to Parkinson'sdisease, idiopathic Parkinson's disease, familiar Parkinson's Disease,Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer'sdisease (LBV), Combined Alzheimer's and Parkinson's disease, pureautonomic failure or multiple system atrophy, comprising: (a) assayingthe existence of alpha-synuclein species and fragments in cells ortissue samples of a subject using one or more antibodies thatspecifically bind to alpha-synuclein; and (b) comparing the level of theantigen with a control level, e.g. levels in normal tissue samples,whereby an increase in the assayed level of antigen compared to thecontrol level of antigen is indicative of the disease or disorder, orindicative of the severity of the disease or disorder.

Antibodies of the invention can be used to assay alpha-synucleinmonomer, oligomers, fibrillary forms or fragments of alpha-synuclein ina biological sample using immunohistochemical methods well-known in theart. Other antibody-based methods useful for detecting protein includeimmunoassays such as the enzyme linked immunoassay (ELISA),radioimmunoassay (RIA) and mesoscale discovery platform based assays(MSD). Suitable antibody labels may be used in such kits and methods,and labels known in the art include enzyme labels, such as alkalinephosphatase and glucose oxidase; radioisotope labels, such as iodine(¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (^(99m)Tc); and luminescent labels, such as luminol andluciferase; and fluorescent labels, such as fluorescein and rhodamine.

The presence of labeled anti-alpha-synuclein antibodies or theiralpha-synuclein-binding fragments may be detected in vivo for diagnosispurposes. In one embodiment, diagnosis comprises: a) administering to asubject an effective amount of such labeled molecule; b) waiting for atime interval following administration to allow the labeled molecule toconcentrate at sites (if any) of Aβ deposition and to allow for unboundlabeled molecule to be cleared to background level; c) determining abackground level; and d) detecting the labeled molecule in the subject,such that detection of labeled molecule above the background level isindicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the molecule is labeled with an imaging moietysuitable for detection using a particular imaging system known to thoseskilled in the art. Background levels may be determined by variousmethods known in the art, including comparing the amount of labeledantibody detected to a standard value previously determined for aparticular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

In a further aspect, the invention relates to an antibody orantigen-binding fragments thereof, of the invention, for use inmedicine.

In a further aspect, the invention relates to an antibody orantigen-binding fragments thereof, of the invention, for use intreating, diagnosing or imaging a synucleinopathy.

In one embodiment, the monoclonal antibody, or antigen-binding fragmentthereof, is for use in treating Parkinson's disease, idiopathicParkinson's disease, familiar forms of Parkinson's Disease, Diffuse LewyBody Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),Combined Alzheimer's and

Parkinson's disease, pure autonomic failure or multiple system atrophy.

In a further aspect, the invention relates to the use of the antibody,or antigen-binding fragment thereof, of the invention, in themanufacture of a medicament for treating, diagnosing or imaging asynucleinopathy.

In a further aspect, the invention relates to a treating, diagnosing orimaging Parkinson's disease or other synucleinopathies, comprisingadministering an effective dosage of an antibody of the invention, or anantigen-binding fragment thereof.

Preferably, in the uses and methods of those aspects of the invention,the treatment is chronic, and is preferably for at least 2 weeks, suchas at least for 1 month, 6, months, 1 year or more.

In a further aspect, the invention provides a kit comprising theantibody, or antigen-binding fragment thereof, of the invention.

SEQ ID NO: 1 GM37 CDR 1 Heavy Chain SEQ ID NO: 2 GM37 CDR 2 Heavy ChainSEQ ID NO: 3 GM37 CDR 3 Heavy Chain SEQ ID NO: 4 GM37 CDR 1 Light ChainSEQ ID NO: 5 GM37 CDR 2 Light Chain SEQ ID NO: 6 GM37 CDR 3 Light ChainSEQ ID NO: 7 GM37 Heavy Chain Variable Domain SEQ ID NO: 8 GM37 LightChain Variable Domain SEQ ID NO: 9 Epitope 112-117 of HumanAlpha-Synuclein SEQ ID NO: 10 Human Alpha-Synuclein SEQ ID NO: 11A-Syn-AAKK-BAP SEQ ID NO: 12 A-Syn-BAAK-BAP SEQ ID NO: 13 A-Syn-BBAA-BAPSEQ ID NO: 14 A-Syn-BBKK-BAP SEQ ID NO: 15 A-Syn-120-140_Del-BAP SEQ IDNO: 16 Residues 1-119 of Human Alpha-Synuclein SEQ ID NO: 17 Kappa LightChain Constant domain SEQ ID NO: 18 lgG1 Heavy Chain Constant domain SEQID NO: 19 GM285 Epitope 112-115 SEQ ID NO: 20 GM285 CDR 1 Heavy ChainSEQ ID NO: 21 GM285 CDR 2 Heavy Chain SEQ ID NO: 22 GM285 CDR 3 HeavyChain SEQ ID NO: 23 GM285 CDR 1 Light Chain SEQ ID NO: 24 GM285 CDR 2Light Chain SEQ ID NO: 25 GM285 CDR 3 Light Chain SEQ ID NO: 26 GM285Heavy Chain Variable Domain SEQ ID NO: 27 GM285 Light Chain VariableDomain SEQ ID NO: 28 GM285 lgG1 Heavy Chain Constant domain SEQ ID NO:29 GM285 Kappa Light Chain Constant domain SEQ ID NO: 30 GM37 Variant 1Heavy Chain Variable Domain SEQ ID NO: 31 GM37 Variant 2 Heavy ChainVariable Domain SEQ ID NO: 32 GM37 Variant 3 Heavy Chain Variable DomainSEQ ID NO: 33 GM37 Variant 1 Heavy Chain CDR 2 SEQ ID NO: 34 GM37Variant 2 Heavy Chain CDR 2 SEQ ID NO: 35 GM37 Variant 3 Heavy Chain CDR2 SEQ ID NO: 36 9E4 Binding Epitope SEQ ID NO: 37 Human Beta-SynucleinSEQ ID NO: 38 Human Gamma-Synuclein SEQ ID NO: 39 Alpha-SynucleinOrtholog for Cynomolgus Monkey SEQ ID NO: 40 Alpha-Synuclein Orthologfor Rat SEQ ID NO: 41 Alpha-Synuclein Ortholog for Mouse SEQ ID NO: 429E4 HC SEQ ID NO: 43 9E4 LC

EMBODIMENTS OF THE INVENTION

As would be apparent from the text and the Examples the inventionfurther relates to the below embodiments

-   1. A monoclonal antibody, or antigen-binding fragment thereof    capable of specifically binding to an epitope within amino acids    112-117 on alpha-synuclein (SEQ ID NO:9 (ILEDMP)).-   2. The monoclonal antibody, or antigen-binding fragment thereof,    according to Embodiment 1 which competes with the antibody GM37 for    binding to said epitope.-   3. The monoclonal antibody, or antigen-binding fragment thereof,    according to Embodiment 1, which is GM37, GM37 variant 1, GM37    variant 2 or GM37 variant 3.-   4. A monoclonal antibody, or antigen-binding fragment thereof    capable of specifically binding to an epitope within amino acids    112-115 on alpha-synuclein (SEQ ID NO:19 (ILED)).-   5. The monoclonal antibody, or antigen-binding fragment thereof,    according to Embodiment 1 or 4, which is GM285.-   6. The monoclonal antibody, or antigen-binding fragment thereof,    according to the previous Embodiments, wherein the antibody    comprises or consists of an intact antibody.-   7. The monoclonal antibody, or antigen-binding fragment thereof,    according to any one of the preceding Embodiments comprising or    consisting of an antigen-binding fragment selected from the group    consisting of Fv fragments (e.g. single chain Fv and    disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′    fragments and F(ab)₂ fragments) and domain antibodies (e.g. single    VH variable domains or VL variable domains).-   8. The monoclonal antibody, or antigen-binding fragment thereof,    according to any one of the preceding Embodiments wherein the    monoclonal antibody is selected from the group consisting of    antibodies of subtype IgG1, IgG2, IgG3 and IgG4.-   9. The monoclonal antibody, or antigen-binding fragment thereof,    according to any one of the preceding Embodiments wherein the    antibody or antigen-binding fragment exhibits one or more of the    following properties:    -   (i) a binding affinity (K_(D)) for alpha-synuclein between        0.5-10 nM, such as 1-5 nM or 1-2 nM;    -   (ii) capability of inhibiting protease truncation of        alpha-synuclein fibrils;    -   (iii) capability of reversing impairment in basal synaptic        transmission in F28-snca transgenic mice;    -   (iv) capability of reducing levels of alpha-synuclein in the        mouse hippocampus as measured by in vivo microdialysis;    -   (v) capability, when administered chronically, to restore motor        function in a rat model of Parkinson's disease;    -   (vi) Capability to prevent seeding of alpha-synuclein (such as        accumulation of insoluble phosphorylated alphasynuclein in vitro        and/or in a mouse model of Parkinson's disease); and/or    -   (vii) Capability to bind truncated alpha-synuclein in a human        brain.-   10. The monoclonal antibody, or antigen-binding fragment thereof,    according to any one of the preceding Embodiments that is human or    humanized.-   11. A monoclonal antibody or the monoclonal antibody according to    Embodiments 1-3 and 6-10, or antigen-binding fragment thereof,    comprising a heavy chain variable domain comprising the following    CDRs:    -   a) GFTFSSYAMT (SEQ ID NO:1) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference;    -   b) AIRS(N/S/Q/H) GDRTD YADSVKG (SEQ ID Nos:2, 33, 34, 35) or an        amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference; or    -   c) AKNWAPFDS (SEQ ID NO:3) or an amino acid sequence having with        no more than 4 amino acid differences, or no more than 3 amino        acid differences, or no more than 2 amino acid differences, or        no more than 1 amino acid difference.-   12. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 11 comprising a heavy chain variable domain    comprising the CDRs of SEQ ID NOs:1 and 3 and one of SEQ ID NOs:2    and 33, 34 or 35.-   13. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 11 comprising or consisting of a heavy chain    variable domain selected from the group consisting of:

a) (SEQ ID NO: 7) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQAPGKGLEWVSA IRSNGDRTDY ADSVKGRFTI SRDNSQNTLYLQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS, b) (SEQ ID NO: 30)EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQAPGKGLEWVSA IRSSGDRTDY ADSVKGRFTI SRDNSQNTLYLQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS, c) (SEQ ID NO: 31)EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQAPGKGLEWVSA IRSQGDRTDY ADSVKGRFTI SRDNSQNTLYLQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS, or d) (SEQ ID NO: 32)EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQAPGKGLEWVSA IRSHGDRTDY ADSVKGRFTI SRDNSQNTLYLQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS.

-   14. A monoclonal antibody or the monoclonal antibody according to    Embodiments 1-3 and 6-13, or antigen-binding fragment thereof,    comprising a light chain variable domain comprising the following    CDRs:    -   a) ASQSVSSSYLA (SEQ ID NO:4) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference;    -   b) GASSRAT (SEQ ID NO:5) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference; or    -   c) QQYGSSPWT (SEQ ID NO:6) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference.-   15. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 14 comprising a light chain variable domain    comprising the CDRs of SEQ ID NOs:4, 5 and 6.-   16. The antibody or antigen-binding fragment thereof according to    Embodiment 14 comprising a light chain variable domain comprising or    consisting of the amino acid sequence:

(SEQ ID NO: 8) EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQKPGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLEPEDFAVYYCQ QYGSSPWTFG QGTKVEIK.

-   17. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 14 comprising a light chain comprising or    consisting of the amino acid sequence:

(SEQ ID NO: 8) EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQKPGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLEPEDFAVYYCQ QYGSSPWTFG QGTKVEIK.

-   18. The monoclonal antibody or antigen-binding fragment thereof    according Embodiments 1-3 and 6-17 comprising a light chain variable    domain comprising or consisting of the amino acid sequence of SEQ ID    NO:8 and heavy a chain variable domain comprising or consisting of    the amino acids given in either SEQ ID No:7, 33, 34 or 35.-   19. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiments 1-3 and 6-18 comprising a light chain    comprising or consisting of the amino acid sequence of SEQ ID NO:8    and heavy a chain variable domain comprising or consisting of the    amino acids given in SEQ ID NO:34 having an increased thermal    stability, such as an increased stability to prevent aggregate and    unfold as shown in FIG. 27 , being between 2%-10% more stable at    temperatures above 65° C. compared to GM37 wt, 2%-8% more stable at    temperatures above 65° C. compared to GM37 wt or 2%-5% more stable    at temperatures above 65° C. compared to GM37 wt.-   20. A monoclonal antibody or the monoclonal antibody according to    Embodiments 1-10, or antigen-binding fragment thereof, comprising a    heavy chain variable domain comprising the following CDRs:    -   a) AASGFTFSRFTMT (SEQ ID NO:20) or an amino acid sequence having        no more than 4 amino acid differences, or no more than 3 amino        acid differences, or no more than 2 amino acid differences, or        no more than 1 amino acid difference;    -   b) AISGSGGGTS YADSVKG (SEQ ID NO:21) or an amino acid sequence        having no more than 4 amino acid differences, or no more than 3        amino acid differences, or no more than 2 amino acid        differences, or no more than 1 amino acid difference; or    -   c) AKNWAPFDY (SEQ ID NO:22) or an amino acid sequence having        with no more than 4 amino acid differences, or no more than 3        amino acid differences, or no more than 2 amino acid        differences, or no more than 1 amino acid difference.-   21. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 20 comprising a heavy chain variable domain    comprising the CDRs of SEQ ID NOs:20, 21 and 22.-   22. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 20 comprising a heavy chain variable domain    comprising or consisting of the amino acid sequence

(SEQ ID NO 26) EVQLLESGGG LVQPGGSLRL SCAASGFTFS RFTMTWVRQAPGKGLEWVSA ISGSGGGTSY ADSVKGRLTV SRDNSKNTLYLQMNSLRAED TAVYYCAKNW APFDYWGQGT LVTVSS.

-   23. A monoclonal antibody or the monoclonal antibody according to    any one of Embodiments 1-10 and 20-22, or antigen-binding fragment    thereof, comprising a light chain variable domain comprising the    following CDRs:    -   d) RASQSVSRSYLA (SEQ ID NO:23) or an amino acid sequence having        no more than 4 amino acid differences, or no more than 3 amino        acid differences, or no more than 2 amino acid differences, or        no more than 1 amino acid difference;    -   e) GASSRAT (SEQ ID NO:24) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference; or    -   f) QQYGSSPWT (SEQ ID NO:25) or an amino acid sequence having no        more than 4 amino acid differences, or no more than 3 amino acid        differences, or no more than 2 amino acid differences, or no        more than 1 amino acid difference.-   24. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 23 comprising a light chain variable domain    comprising the CDRs of SEQ ID NOs:23, 24 and 25.-   25. The antibody or antigen-binding fragment thereof according to    Embodiment 24 comprising a light chain variable domain comprising or    consisting of the amino acid sequence of:

(SEQ ID NO: 27) EIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQKPGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTVSRLEPEDFAVYYCQ QYGSSPWTFG QGTKVEIK.

-   26. The monoclonal antibody or antigen-binding fragment thereof    according to any one of the preceding Embodiments comprising a light    chain variable domain comprising or consisting of the amino acid    sequence of SEQ ID NO:27 and heavy a chain variable domain    comprising or consisting of the amino acids given in either SEQ ID    NO:26.-   27. The monoclonal antibody or antigen-binding fragment thereof    according to any one of the preceding Embodiment comprising an Fc    region.-   28. The monoclonal antibody or antigen-binding fragment thereof    according to any one of the preceding Embodiment further comprising    a moiety for increasing the in vivo half-life of the agent.-   29. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 28 wherein the moiety for increasing the in    vivo half-life is selected from the group consisting of polyethylene    glycol (PEG), human serum albumin, glycosylation groups, fatty acids    and dextran.-   30. The monoclonal antibody or antigen-binding fragment thereof    according to any one of the preceding Embodiments wherein the    antibody polypeptide further comprises a detectable moiety.-   31. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 30 wherein the detectable moiety is a    fluorescent label, a chemiluminescent label, a paramagnetic label, a    radioisotopic label or an enzyme label.-   32. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 30 or 31 wherein the detectable moiety    comprises or consists of a radioisotope.-   33. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 32 wherein the radioisotope is selected from    the group consisting of ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr,    ¹²³I and ²⁰¹Tl.-   34. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 30 wherein the detectable moiety comprises    or consists of a paramagnetic isotope.-   35. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 34 wherein the paramagnetic isotope is    selected from the group consisting of ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and    ⁵⁶Fe.-   36. The monoclonal antibody or antigen-binding fragment thereof    according to any of Embodiments 30 to 35 wherein the detectable    moiety is detectable by an imaging technique such as SPECT, PET,    MRI, optical or ultrasound imaging.-   37. The monoclonal antibody or antigen-binding fragment thereof    according to any of Embodiments 30 to 36 wherein the detectable    moiety is joined to the antibody or antigen-binding fragment thereof    indirectly, via a linking moiety.-   38. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 37 wherein the linking moiety is selected    from the group consisting of derivatives of    1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA),    deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic    avid (DTPA), derivatives of    S-2-(4-lsothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic    acid (NOTA) and derivatives of    1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA).-   39. An isolated nucleic acid molecule encoding an antibody or    antigen-binding fragment thereof according to any one of the    preceding Embodiments or a component polypeptide chain thereof.-   40. A nucleic acid molecule according to Embodiment 39 wherein the    molecule is a cDNA molecule.-   41. A nucleic acid molecule according to Embodiment 30 or 31    encoding an antibody heavy chain or variable domain thereof.-   42. A nucleic acid molecule according to any one of Embodiments 39    to 41 encoding an antibody light chain or variable domain thereof.-   43. A vector comprising a nucleic acid molecule according to any one    of Embodiments 39 to 42.-   44. A recombinant host cell comprising a nucleic acid molecule    according to any one of Embodiments 39 to 42 or a vector according    to Embodiment 43.-   45. A method for producing an antibody or antigen-binding fragment    according to any one of the Embodiments 1 to 27, the method    comprising culturing a host cell as defined in Embodiment 44 under    conditions which permit expression of the encoded antibody or    antigen-binding fragment thereof.-   46. A pharmaceutical composition comprising the monoclonal antibody    or antigen-binding fragment according to any one of Embodiments 1 to    35 and a pharmaceutical acceptable carrier.-   47. The monoclonal antibody or antigen-binding fragment thereof of    Embodiments 1-35 for use in medicine.-   48. The monoclonal antibody or antigen-binding fragment thereof of    Embodiments 1-35 for use in treating, diagnosing or imaging a    synucleinopathy.-   49. The monoclonal antibody or antigen-binding fragment thereof    according to Embodiment 48 for use in treating Parkinson's disease    (including idiopathic and inherited forms of Parkinson's disease),    Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body    variant of Alzheimer's disease (LBV), Combined Alzheimer's and    Parkinson disease, pure autonomic failure and multiple system    atrophy.-   50. Use of a monoclonal antibody or antigen-binding fragment thereof    of Embodiments 1-35 in the manufacturing of a medicament for    treating, diagnosing or imaging a synucleinopathy.-   51. The use of a monoclonal antibody or antigen-binding fragment    thereof according to Embodiment 50 in the manufacturing of a    medicament for treating Parkinson's disease (including idiopathic    and inherited forms of Parkinson's disease Parkinson's disease),    Gaucher's Disease, Diffuse Lewy Body Disease (DLBD), Lewy body    variant of Alzheimer's disease (LBV), Combined Alzheimer's and    Parkinson disease, pure autonomic failure and multiple system    atrophy.-   52. A method of treating, diagnosing or imaging a synucleinopathy in    a subject, said method comprising administering the pharmaceutical    composition of Embodiment 46 to said subject in an effective amount.-   53. The antibody, or antigen-binding fragment thereof, for use    according to Embodiment 48, or the use according to Embodiment 50,    or the method according to Embodiment 52 for treating Parkinson's    disease (including idiopathic and inherited forms of Parkinson's    disease Parkinson's disease), Gaucher's Disease, Diffuse Lewy Body    Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),    Combined Alzheimer's and Parkinson disease, pure autonomic failure    and multiple system atrophy.-   54. The antibody, or antigen-binding fragment thereof, for use; or    the use; or the method according to Embodiment 52 or 53, wherein the    treatment is chronic-   55. The antibody, or antigen-binding fragment thereof, for use; or    the use; or the method according to Embodiment 52, wherein the    chronic treatment is for at least 2 weeks, such as at least for 1    month, 6, months, 1 year or more.-   56. The antibody, or antigen-binding fragment thereof, for use; or    the use; or the method according to any one of Embodiments 47 to 55,    wherein the subject is human.-   57. A kit comprising the antibody or antigen-binding fragment    thereof according to Embodiments 1-35-   58. The kit according to Embodiment 57 for use in medicine-   59. The monoclonal antibody or antigen-binding fragment thereof of    Embodiments 30-35 for use in detecting or measuring the presence or    amount of said alpha-synuclein in the brain or a body fluid of a    subject.-   60. The monoclonal antibody or antigen-binding fragment thereof of    Embodiments 59, wherein said detection or measurement comprises in    vivo imaging of said anti-synuclein antibody bound to said    alpha-synuclein.-   61. The monoclonal antibody or antigen-binding fragment thereof of    Embodiments 30-35 wherein said detection or measurement comprises ex    vivo imaging of said anti-synuclein antibody bound to said    alpha-synuclein.

EXAMPLES Example 1: Antibody Screening

1. Immunogen and Ligand Production

The following proteins were acquired or produced for use as immunogensshown in FIG. 1 . The mice were immunized with three immunogens: fulllength recombinant human alpha-synuclein fibrils; human alpha-synucleinrecombinant protein containing amino acids 1-60 (Rpeptide, Bogart, Ga.)and human alpha-synuclein recombinant protein containing amino acids1-119. To make the fibrils from the full length the alpha-synuclein alyophilized product from Rpeptide, Bogart, Ga. (Catalog number S-1001-2)was used. This was dissolved in 20 mM tris and 300 mM NaCl buffer atconcentration of 1 mg/ml protein. To make the fibrils the proteinsolution was incubated 170 μl aliquots in 96 well plate with a 70 μmdiameter ceramic bead in each well at 200 rpm in Vortemp 56 shakerincubator (Labnet International, Edison, N.J., USA), at 37° C. for 7days, and the formation of fibrils was followed by adding thioflavin Tand measuring fluorescence in one of the wells. The recombinantalpha-synuclein containing amino acids 1-60 was dissolved in water togive a concentration of 1 mg/ml.

The recombinant alpha-synuclein containing amino acids 1-119 was madeusing the following construct: A synthetic gene coding for a 6 aminoacid Histidine tag, followed by factor Xa cleavage site and sequencecoding for human alpha-synuclein amino acids 1-119:

(SEQ ID NO: 16) MAHHHHHHIE GRMDVFMKGL SKAKEGVVAA AEKTKQGVAEAAGKTKEGVL YVGSKTKEGV VHGVATVAEK TKEQVTNVGGAVVTGVTAVA QKTVEGAGSI AAATGFVKKD QLGKNEEGAP QEGILEDMPV Dwas synthezised by Genscript and cloned into Ndel-XhoI site in pET24a(+)expression vector (Novagen).

The expression vector was transformed into E. coli BL21 and a singlecolony picked for expression using the overnight express autoinductionsystem from Novagen (User protocol TB383 rev. H1005). The scale was 500ml of final culture volume. Cells were harvested by centrifugation 10min at 6000 g and subsequently lyzed using BugBuster protein extractionReagent (User protocol TB245 Rev. 0304). After lysis the sample wascleared by centrifugation and the supernatant used for furtherpurification.

The His-tagged protein was purified on a 5 ml HisTrap column (GEhealthcare) equilibrated in 20 mM Sodium phosphate pH7.5, 1 M NaCl(A-buffer). After sample application and wash using A-buffer the proteinwas eluted in a gradient to 0.25 M Imidazole in A-buffer over 20 columnvolumes. Fractions of 5 ml were collected and analyzed by SDS-PAGE.Fractions with the protein of interest was pooled, concentrated andapplied to an S200 (26/60) size exclusion column (GE healthcare) in 10mM tris pH 7.4, 300 mM NaCl. Again fractions were pooled according topresence in SDS-PAGE of a band with expected size.

To remove the N-terminal tag, the purified his-tagged alpha-synuclein1-119 was incubated with factor Xa in a 1:50 ration using the Novagenkit (69037-3FRX). After overnight incubation, the factor Xa was removedbatchwise using Xarrest agarose. The cleaved alpha-synuclein 1-119 wasfinally purified by permissive HisTrap chromatography as describedabove. From the flow through the purified alpha-synuclein 1-119 wasobtained and concentrated to ˜400 μg/ml using centricon concentrationdevises.

Alpha-synuclein (Rpeptide) was rehydrated in PBS at 2 mg/ml andperoxynitirite (100 μL/mg protein) was added dropwise while mixing. Thenitrosylated alpha-synuclein was then dialyzed in 5 L PBS and stored at−20° C.

Dopamine was used to oxidize alpha-synuclein. Equal volumes of a 200 uMsolution of Dopamine-HCL (Sigma P5244) prepared in 10 mM PBS, pH7.4 anda 28 μM solution of alpha-synuclein (Rpeptide) in 10 mM PBS, pH7.4 werecombined. The resulting 14 uM alpha-synuclein/100 uM Dopamine wereincubated at 37° C. O/N (over night). The oxidized alpha-synuclein wasthen dialyzed in PBS and stored at −20° C.

Different native and chimeric versions of synuclein proteins wereproduced in order to screen a diverse library of anti-alpha-synucleinantibodies. Screening constructs included the following: human, mouse,rat and cynomolgus monkey alpha-synuclein, human Beta-synuclein, HumanGamma-synuclein (FIGS. 21 and 22 ) and lastly an alpha-synucleinderivative that lacked residues 120-140 of alpha-synuclein. In addition,a series of 4 shuffle constructs: A-Syn-AAKK-BAP, A-Syn-BAAK-BAP,A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID Nos:11-14) were produced. Theseconstructs contained linear stretches of human alpha-synuclein (A),human Beta-synuclein (B) and chicken alpha-synuclein (K). Gene werecloned containing a Biotin Acceptor Peptide (BAP) tag fused to theC-terminus of the ligands in order to facilitate site specificbiotinylation of each of the ligands. The bioytinylation allowed forattachment of the ligands to beads used in the soluble ELISA format.Mammalian expression vectors were constructed carrying the differentalpha-synuclein BAP tag fusion constructs (ASynBAP). The ligands wereexpressed in HEK 293 cells using transient transfection (Genmab A/S).

2. Immunization

Antibodies HuMab-Synuclein were derived from the immunizations of HuMAbmouse strain HCo17-BALB/c and HCo12-BALB/c mice, double knock out forthe mouse immunoglobulin (Ig) heavy and mouse kappa light chain, whichprevents the expression of antibodies that are completely murine (humanmonoclonal antibody; Medarex Inc., San Jose, Calif., USA). The variousmouse strains were made transgenic by the insertion of human Ig heavyand human Ig kappa light chain loci and differ in the number of human VH(variable domain of heavy chain) and VL (variable domain of light chain)genes.

48 Mice were immunized alternating intraperitoneally (IP) with 20 μgantigens and subcutaneously (SC, at the tailbase) with the sameimmunogen, with an interval of 14 days. A maximum of eight immunizationswere performed, 4 IP and 4 SC.

The first immunization was performed with alpha-synuclein immunogens incomplete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Mich.,USA), the following immunizations in incomplete Freund's adjuvant (IFA).When serum titers were found to be sufficient (dilution of serum of 1/50or lower found positive in antigen specific screening assay as describedin herein above on at least two sequential, biweekly, screening events),mice were additionally boosted twice intravenously (IV) with 10 μgalpha-synuclein immunogen protein in 100 μL PBS, four and three daysbefore fusion.

The immunization protocols are shown in FIG. 1 .

Antibody 37 came from an immunization protocol where human full lengthα-Synuclein-fibrils was used, alternating with alpha-synucleinC-terminally truncated forms with amino acids 1-60 and 1-119.

Antibody 285 came from an immunization protocol where Humanα-Synuclein-monomer 1-140 was used for the first 4 immunizations. Ifthere was no titer, the immunization was continued with fibrils (ip/sc),otherwise it was continued with monomer.

3. HuMab Hybridoma Generation

HuMAb mice with sufficient antigen-specific titer development as definedabove were sacrificed and the spleen and lymph nodes flanking theabdominal aorta and caval vein were collected. Fusion of splenocytes andlymph node cells with a mouse myeloma cell line was done byelectrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences,Glen Burnie, Md., USA), essentially according to the manufacturer'sinstructions. Fused cells were seeded in fusion medium containing 10%Fetal Clone I Bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5U/mL penicillin, 0.5 U/mL streptomycin (Cambrex), 50 μM2-mercaptoethanol (Invitrogen), 600 ng/mL interleukin 6 (IL-6)(Strathmann), 1×HAT (Sigma) and 0.5 mg/mL kanamycin (Invitrogen) in HyQmADCF-Mab (Perbio). After ten days, supernatant was harvested and cellswere refreshed with harvest medium, containing 10% Fetal Clone I Bovineserum, 0.5 U/mL penicillin, 0.5 U/mL streptomycin, 600 ng/mL IL-6 and1×proHT (Cambrex) in HyQ mADCF-Mab. Supernatants of the hybridomacultures were screened by primary screening assays. Supernatants werecharacterized for binding to eight different ligands. These included 4orthologs: human, mouse, rat and cynomologus monkey, humanalpha-synuclein Beta-synuclein and human Gamma-synuclein (SEQ ID NOs37-41) and lastly they were tested for their ability to bind to a humanalpha-synuclein derivative that lacked residues 120-140 ofalpha-synuclein.

The screening of anti-alpha-synuclein antibodies was performed using ahigh throughput suspension ELISA format using automated liquid handlingsystems (Genmab A/S). The reading of the plates was performed by twosystems, the FMAT 8200 from Applied Biosystems was used to read 384 wellplates and the ImageXpress Velos Cytometer from Molecular Devices wasused to read the 1536 well plates.

In the primary screen clones were characterized by their ability to bind8 different ligands. These included a series of 4 shuffle constructs:A-Syn-AAKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ IDNOs:11-14), alpha-synuclein 120-140 deletion-BAP, nitrated humanalpha-synuclein-BAP and oxidized human alpha-synuclein-BAP.

In short, the sera or supernatant potentially containing alpha-synucleinspecific antibodies were added to the beads to allow binding toalpha-Synuclein and/or alpha-synuclein derived constructs. The bindingof the anti-alpha-synuclein antibodies is detected using a fluorescentconjugate, DyLight649 conjugated goat antihuman IgG, Fc specific. Twoknown mouse anti-alpha-synuclein antibodies, LB509 and Syn211, wereincluded in screenings as positive controls. To ensure specificdetection of alpha-synuclein antibodies, an anti-alpha-synuclein serapool is used as a negative control in the 384 well format titerscreening while human ChromPure IgG is used in the 1536 well format8-bead based assay.

Hybridoma cells from the best primary wells were seeded in semisolidmedium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2×complete medium (Hyclone, Waltham, USA). For each primary well, a wellof a Genetix black 6-well plate was seeded. From each well, 25 subclones were picked, using the ClonePix system (Genetix). The sub cloneswere picked in harvest medium. After seven days, the supernatants of thesub clones were screened again for Synuclein-specific human IgG bindingand the human IgG concentration was measured using Octet (Fortebio,Menlo Park, USA). From each primary well, the best sub clone wasselected and expanded in expansion medium containing only 600 ng/mLIL-6, 0.5 U/mL penicillin, 0.5 U/mL streptomycin and 1×proHT. The subclones were expanded from one 96-well plate well to one 24-well platewell to four 24-well plate wells to six 6-well plate wells. Clonesderived by this process were designated as primary clones (PC).

Additional antibody binding studies were performed using Octet 384RED(Fortebio, Menlo Park, USA). HuMab antibody solutions of 2 μg/ml weremade by dilution in sample diluent (ForteBio, art. No. 18-5028). Aminereactive sensors (ForteBio, art.no. 18-0008) were used forimmobilization of HuMabs. Prior to coupling to amine reactive sensors,HuMabs were diluted in MES pH 6.0 buffer (18-5027). Coupling wasperformed at 30° C. and 1000 rpm as follows: Amine reactive sensors werepre-wet in PBS and subsequently activated with EDC/NHS (ForteBio.Art.no. 18-1033/18-1034) activation solution (according tomanufacturer's instruction) for 300 seconds. Activated sensors wereimmobilized with HuMabs during 600 seconds.

The binding of 37 and 285 in Octet to recombinant human, cynomolgus andmouse alpha-synuclein, and lack of binding to human beta orgamma-synuclein is shown in FIG. 2

4. Sequence Analysis of the Synuclein-Specific HuMab Variable Domainsand Cloning in Expression Vectors

Total RNA was prepared from 0.2 to 5×106 hybridoma cells and5′-RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA,using the SMART RACE cDNA Amplification kit (Clontech), according to themanufacturer's instructions. VH and VL coding regions were amplified byPCR and cloned directly, in frame, in the p33G1f and p33Kappa expressionvectors (containing the human IgG1/kappa constant domain encodingsequences), by ligation independent cloning (Aslanidis, C. and P. J. deJong, Nucleic Acids Res 1990; 18(20): 6069-74). For each antibody, 16 VLclones and 16 VH clones were sequenced. Clones with a correct OpenReading Frame (ORF) were selected for further study and expression.Vectors of all combinations of heavy chains and light chains weretransiently co-expressed in Freestyle™ 293-F cells using 293fectin.

In the case of GM37 sequencing of the VH region identified an extracysteine in the CDR3 domain at position 106. In order to eliminate thepossibility of misfolding and potential loss of antibody activity due todisulfide bond formation the cysteine was mutated to serine at position106.

Comparator antibody 9E4 was generated based on the VH and VL sequencederived from hybridoma PTA-8221 (US patent 20080175838) (SEQ ID NO 42and 43)

5. Expression/Purification of Antibodies

Antibodies were produced by transfection in HEK293 6E cells using thepTT5 vectors and PElpro as a transient transfection agent (NationalResearch Council of Canada). In short, The heavy and light chains weretransfected into HEK293 cells using PEIpro (VWR), and cells weresupplemented with TN1 (Sigma) 24 hours after transfection. Cells weregrown until the viability approached 50%, and yield of antibody measuredby easy IgG titre (Thermo). Culture supernatant was filtered over 0.2 μmdead-end filters, loaded on 5 mL Protein A columns (rProtein A FF,Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. Theeluate was immediately neutralized with 2M Tris-HCl, pH 9 and dialyzedto 12.6 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 (B.Braun), 0/N. After dialysis,samples were sterile-filtered over 0.2 μm dead-end filters. Purity wasdetermined by SDS-PAGE and concentration was measured by nephelometryand absorbance at 280 nm. Purified antibodies were aliquoted and storedat −80° C.

Example 2: Antibody Characterization Using Surface Plasmon Resonance

Real time binding of the antibodies to alpha-synuclein was measuredusing a BIAcore® 3000. A capture surface was prepared by amine-couplinga polyclonal rabbit Anti-Mouse antibody (part of Mouse Antibody CaptureKit, GE Healthcare, Cat. no: BR-1008-38) in first flow cell (Fc1) andsecond flow cell (Fc2) of a CM5 chip (BIAcore®). The mouse antibody wascaptured in Fc2 at the concentration required to achieve a ligand levelof around 500RU. The baseline was allowed to stabilize for 10 min beforeinjecting analyte (ASynBAP) in Fc1-2 at 30 μl/min. ASynBAP was run at100-3200 nM and 25-3200RU, respectively. The highest concentration ineach titration series was run in duplicate. The surface was regeneratedwith 10 mM Glycine-HCl, pH 1.7 (30 sec inject) to remove captured mouseantibody and analyte in the end of each cycle. HBS-EP (GE Healthcare,Cat. No: BR-1001-88) was used as running buffer and sample diluent inall experiments and the assay was run at 25° C. All samples were kept at4° C. before acquisition.

The response recorded in Fc1, where capture antibody had beenimmobilized but no Alpha-Synuclein antibody captured, was subtractedfrom the response in Fc2. A 1:1 or 2:1 binding algorithm was fit to thedataset using BIAevaluation software version 4.1.1. Results can be seenin FIGS. 3, 4 and 5 showing binding of antibody 37, 285 and 9E4 to humanalpha-synuclein.

Example 3: Epitope Mapping

Epitope mapping of the antibodies to alpha-synuclein was done witharrays of overlapping linear peptides at Pepscan (Pepscan Zuidersluisweg2 8243 RC Lelystad, The Netherlands). The binding of antibody to each ofthe synthesized 20 mer peptides was tested in a Pepscan based ELISA. Thelinear peptide array covering the entire coding sequence ofalpha-synuclein, as well as all peptides with oxidized methionines ornitrosylated tyrosines, were incubated with primary antibody solution(overnight at 4° C.). After washing, the peptide arrays were incubatedwith a 1/1000 dilution of an antibody peroxidase conjugate (SBA, cat.nr. 2010-05) for one hour at 25° C. After washing, the peroxidasesubstrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2μl/ml of 3 percent H₂O₂ were added. After one hour, the colordevelopment was measured. The color development was quantified with acharge coupled device (CCD)—camera and an image processing system. Fordata processing the values were obtained from the CCD camera range from0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. Theresults were quantified and stored into the Peplab database.Occasionally a well contains an air-bubble resulting in a false-positivevalue, the cards are manually inspected and any values caused by anair-bubble are scored as 0. The binding data of antibody 37 and 285 topeptides containing the sequence ILEDMP or ILED respectively can be seenin FIG. 7 .

Example 4: Immunoprecipitation of Alpha-Synuclein from Human BrainHomogenates of Cingulate Cortex from Patients with Dementia with LewyBodies

The ability of the antibodies to bind to and pull down alpha-synucleinfrom crude homogenates of cingulate cortex from human DLB or healthycontrol (marked with *) was analyzed by immunoprecipitation. Frozensample from human cingulate cortex (obtained throughTissue SolutionsLtd, Scotland) was dissected in cryostat, and 100 mg sample was added to1600 μl cellytic M cell lysis reagent (Sigma C2978) containing proteaseinhibitors and phosphatase inhibitors (Roche). Brain tissue washomogenized until the sample is dissolved completely using Precellysbead homogenizer (Bertin technologies, France) 4×30 sec at 5000 rpm. Thesolution was centrifuged at 3000×g and the supernatant was used as thecrude homogenate for immunoprecipitation.

For immunoprecipitation 10 μg of antibody was mixed with magneticDynabeads protein G beads using manufacturer's instructions (LifeTechnologies, Paisley, UK). The crude brain homogenate was diluted 30fold in lysis buffer (Sigma). Antibody coupled dynabeads were mixed with500 ul of diluted homogenate and incubated 90 minutes at roomtemperature under continuous mixing in a rotator. After incubation thebeads were washed in washing buffer and the bound antigens were elutedusing the non-denaturing elution buffer according to manufacturer'sinstructions (Dynabeads G protocol, Life Technologies, Paisley UK). Theyield of the immunoprecipitation was visualized by Western blotting withdetection mouse monoclonal anti-human alpha-synuclein antibody, (4612,Thermo Scientific). The patterns of bands representing differentmolecular weight forms of alpha-synuclein being pulled down differbetween the 37, 37 variant 2 and 285 antibodies and the comparatorantibody 9E4 in that the 37, 37v2 and 285 antibody can immunoprecipitatethe major alpha-synuclein species, the full length alpha-synuclein (FLasyn 1-140) and the C-terminal terminal truncated species (1-135 and1-119/122), while antibody 9E4 cannot immunoprecipitate the truncatedspecies 1-119/122. FIG. 9 .

Example 5: Inhibition of Protease Truncation of Alpha-Synuclein Fibrilsby Antibodies in Cell Culture

Recombinant alpha-synuclein monomers and fibrils can be taken up byprimary neurons in culture. As shown schematically in FIG. 10 , afteruptake of the alpha-synuclein in neurons, it can be processed byintracellular proteases, such as Calpain I, with the major proteasesensitive site at amino acid 119/122. To investigate truncation ofalpha-synuclein by proteases mouse primary cortical neurons wereprepared as described in Elvang et al. 2009 (Elvang et al. J Neurochem.2009; 110(5):1377-87) and treated with cytarabine on DIV3 (3 days invitro) to inhibit astrocyte growth. On DIV4 (4 days in vitro), theneurons were treated with sonicated (5 min at 50% power in cup hornsonicator) pre-formed alpha-synuclein fibrils (PFFs) at an endconcentration of 0.7 μM alone or together with antibodies in theindicated concentrations. After 24 hours of incubation, the media washarvested and the cells were lysed. Western Blots was run on both mediaand cell lysate using the 4612 antibody (FIG. 11A) (Pierce MA1-90346)and a secondary anti-mouse antibody. After probing with 4612+anti-mouse,the blots were stripped and reprobed with an anti-human-IgG antibody. Onblot with 4612, it can be seen that in the media from cells treated withPFFs only, there were strong bands at 14 and 12 kDa, where 14 kDarepresents the full-length alpha-synuclein (FL-asyn) and 12 kDarepresents the C-terminally truncated fragment 1-119/122 (CT-asyn). Inaddition to that, there were higher molecular weight bands, most likelyrepresenting SDS-resistant oligomeric species. Co-treatment with theisotype control antibody B12 did not change this pattern of proteolysisor uptake.

In the media from cells treated with fibrils together with 37 there wasmainly full length alpha-synuclein (14 kD) and only small amounts of theterminally truncated band (12 kD). In cell lysate inform cells treatedwith fibrils together with 37 there was full length alpha-synucleinonly, indicating that 37 prevent cleavage of FL-alpha-synuclein.Furthermore the total amount of FL-alpha-synuclein is reduced inrelation to cells treated with PFFs only or B12 control antibody. It hasbeen shown by several groups (Games et al, Am J of Pathol, Vol. 182, No.3, March, 2013; Ritchie et al, Health, Vol. 4, Special Issue, 1167-1177,2012; Mishizen-Eberz, Biochemistry, 2005, 44, 7818-7829; Dufty et al, AmJ of Pathol, Vol. 170, No. 5, May 2007) that alpha-synuclein can becleaved by Calpain-1. The cleavage site of Calpain-1 for fibrillizedalpha-synuclein has been found to be in the region 114-122(Mishizen-Eberz, J of Neurochem, 86, 836-847, 2003). In vivo intransgenic animals and human brain 1-119/122 seems to be the maincleavage product—in alpha-synuclein, the cleavage is likely afterasparte 119 or asparagine 122, which is deamidated to aspartate and iscleaved by Calpain or another protease with similar cleavagespecificity. These results indicate that antibody 37 is able to inhibitC-terminal truncation of alpha-synuclein. The epitope of antibody 37overlaps with the enzyme Calpain-1 binding site, so binding of 37 toalpha-synuclein could directly inhibit binding and cleavage mediated byCalpain-1 (FIGS. 10 and 11 ).

The epitope of 285 overlaps with the epitope of 37 and it would also beexpected to inhibit the protease cleavage. The amino acid sequence of37v2 only differs from 37 at one amino acid in CDR and has similarbinding as 37, so it would also be expected to inhibit protease cleavagein similar manner to 37. To investigate if the effect of the antibodieswere dose-dependent, a 24-hour experiment with co-addition of PFFs andantibodies to primary cortical neurons was set up. The concentration ofPFFs were stable (10 μg/ml), whereas the concentrations of controlantibody B12, and antibodies 37, 37v2 and 285 that was tested was 10, 5,1 and 0,1 ug/ml. Alpha-synuclein on Western Blots were detected with1904/4612 antibody (Abcam), which has an epitope in the region 103-108and therefore binds to both the FL and the C-terminally truncatedalpha-synuclein (FIG. 11B). As can be seen from FIG. 11B, both GM37,37v2 and GM285 have a dose dependent inhibition of protease cleavage,with almost complete inhibition of the cleavage at high concentration ofthe antibody.

Example 6: Antibody-Mediated Inhibition of Seeding of Alpha-SynucleinAggregation in Cell Culture

Several studies have shown that exogenous addition of recombinant alphasynuclein fibrillar aggregates can enter cells and recruit endogenousalpha-synuclein and induce alpha-synuclein aggregation andphosphorylation in vitro and in vivo, which resemble LB.(Volpicelli-Daley et al. 2011, Luk et al. 2012a, Luk et al. 2012b,Recasens et al. 2013, Peelaerts et al. 2015). To study seeding ofendogenous mouse alpha-synuclein by recombinant alpha-synuclein seeds,mouse primary cortical neurons prepared as above are plated in 96 wellplates (15,000 cells per well). On day 5 in vitro culture (DIV), 50% ofmedia is changed and supplemented with cytosine arabinoside (final conc.of 1 uM). On DIV 6, half of the media is changed with glia conditionedmedia along with alpha synuclein fibrillary material, either crudefibril seeds or pure seeds. The crude fibril seeds are made fromrecombinant monomeric human alpha-synuclein, which was isolated frombacteria and the monomers were filtered through an Amicon Ultra 100.000cut off filter (Millipore cat. No UFC510096) and adjusted toconcentration of 1 mg/ml in PBS, pH 7.4. To make fibril crude seeds, themonomer solution was incubated in thermomixer at 37 C with continuousmixing (800 rpm) until plateu is reached (evaluated by daily measureswith Thioflavin S). To minimize evaporation a drop of mineral oil wasadded to cover the solution. The total time for incubation was 5-7 days,The pure seeds are made from crude fibril seeds that are centrifuged topurify them and the aggregated pellet is resuspended in fresh PBS andsonicated. The antibodies are added once on DIV 6 along withalpha-synuclein crude seeds. Half of the media in the primary neurons isreplaced with glia conditioned media every week to maintain them up toDIV21. The neurons are fixed and stained for Phospho-synuclein using arabbit antibody specific for phosphorylation of alpha-synuclein at aminoacid S129 (abcam 51253), followed by a fluorescently labelledanti-rabbit antibody, fluorescence is quantified using automatedfluorescent microscopy, Cellomics Arrayscan. Nuclei were detected in onechannel and defined the number of valid cells. Phosphorylatedalpha-synuclein spots were detected in another channel in a pre-definedring-formed area surrounding the nucleus, thus representing thecytoplasm of the cells. The average number of spots per cell wascalculated. Example of cell staining is shown in FIG. 12A.Phosphorylated alpha synuclein spots are not seen in untreated neurons.Neurons incubated with crude or pure seeds (1-10 ng per well) inducephosphorylation of alpha synuclein (FIG. 12A). In neurites,phosphorylated synuclein appears as spots or punctate and some of thephospho-synuclein in the neurites appear elongated.

For fractionation studies cells were harvested in phosphate bufferedsaline solution (PBS) and centrifuged. Pellet was resuspended in 1%triton buffer with protease inhibitors. Samples were kept on ice for 15min. followed by sonication. The samples were centrifuged at 100,000×gfor 30 min. at 4 C. The supernatant is collected and labelled as solublefraction. The pellet was washed once in triton buffer and re-suspendedin 1% SDS buffer followed by sonication. Samples were centrifuged againat 100,000×g for 30 min. The supernatant is collected as insolublefraction. The protein concentrations were measured and samples were runon 4-12% SDS_PAGE gel, blotted on membranes and alpha synuclein andphosphorylated alpha synuclein (S129P) are detected by 4612/1904antibody (Thermo scientific: MA1-90346-human synuclein), S129P-asynantibody (abcam 51253) and mouse synuclein antibody (cellsignalling-D37A6), respectively.

FIG. 12 B shows the Western blot of the soluble and insoluble fractionfrom the primary neurons with and without crude seeds. As can be seenfrom FIG. 12B the addition of the seeds lead to accumulation ofendogenous mouse alpha-synuclein and p-S129-alpha-synuclein andmultimers of phosphorylated mouse alpha-synuclein in the insolublefraction of the cells.

To test if antibodies can inhibit seeding, alpha synuclein synucleinseeds were used at conc. of 6.6 nM (10 ng/well). Different concentrationof antibody and alpha-synuclein seeds were added together on DIV 6, tomake a dose response (starting from highest antibody conc. at 133 nMdown to 133 pM). The neurons were again fixed and stained forPhospho-synuclein (abcam 51253) and fluorescence from cells wasquantified using automated fluorescent microscopy, Cellomics arrayscan.The spots/puncta per cell were counted in Cellomics arrayscan. As can beseen from FIG. 12C, both antibody 37, 37v2 and antibody 285 reducedalpha synuclein phosphorylation in neurons in a dose dependent mannerwith similar maximal inhibition for 37, 37v2 and 285 (around 70-75%) andEC50 around 5 nM. Fractionation of the cellular proteins to soluble andinsoluble fraction after treatment with antibody at the highestconcentration (133 nM) shows that both antibodies 37, 37v2 and 285inhibited the truncation of the recombinant crude seeds and accumulationof C terminally truncated fragment (CT a-syn), and reduced theaccumulation of phosphorylated endogenous mouse alpha-synuclein andaggregated forms of mouse alpha-synuclein in the insoluble fraction, asseen in FIG. 12D.

Example 7. Acute Electrophysiological Effects of Alpha-SynucleinAntibodies In Vivo

High expression levels of human alpha-synuclein are present in thehippocampus of F28-snca transgenic mice, a model overexpressing wildtypealpha-synuclein under the control of the mouse alpha-synuclein promotor(Westerlund M, et al. Mol Cell Neurosci. 2008 December; 39 (4):586-91).Assessment of synaptic transmission and plasticity in the CA1 area ofthe hippocampus in 4 to 6 months old male F28-snca transgenic andage-matched control mice was performed by in vivo electrophysiology. Thedata shows that basal synaptic transmission is significantly impaired inF28-snca transgenic compared to age-matched control mice (FIG. 13 ).

F28-snca transgenic and age-matched control male mice (CRO breeding,Taconic Europe A/S) aged 4 to 6 months were single-housed in controlledtemperature (22±1.5° C.) and humidity conditions (55-65%) and kept in a12:12 hour light/dark cycle (lights on at 06:00 h). Food and water wereavailable ad libitum.

Animals were anesthetized with an intraperitoneal (i.p.) injection ofurethane (1.2 g/kg). Mice were then mounted in a stereotaxic frame,their temperature adjusted to 37.5° C. via a heating pad, and the skullwas exposed. A platinum wire was placed in the frontal bone to act as areference, and an additional hole was drilled for insertion of therecording and stimulating electrodes in the hippocampus, at thefollowing coordinates according to the atlas of Paxinos and Franklin(Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates, 4thEdition, 2001): recording, 1.5-1.7 mm posterior to Bregma, 1.0-1.2 mmlateral to the midline, 1.4-1.7 mm below the surface of the brain;stimulation, 1.8-2.0 mm posterior to Bregma, 1.5-1.7 mm lateral to themidline, 1.5-1.7 mm below the surface of the brain. Animals were left inthe stereotaxic frame through the whole duration of the recordings andtheir level of anesthesia was regularly checked.

Field potentials (fEPSP) were evoked in the CA1 by electricalstimulation of the Schaffer collateral every 30 s, and the depth of therecording electrode was adjusted until a negative fEPSP was recorded inresponse to a unipolar square pulse. The slope of the evoked fEPSP wasmeasured between 30 and 70% of the maximum amplitude of the fEPSP.

Once an optimal fEPSP was induced, basal synaptic transmission wasassessed by the relationship between stimulation intensity and slope ofthe evoked fEPSP (input-output relationship). The different intensitiesof stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA,and were applied successively in increasing order, with 2 to 3 repeatsfor each intensity. Basal synaptic transmission was found to besignificantly impaired in F28-snca transgenic compared to age-matchedcontrol mice.

The identified impairments in basal synaptic transmission in F28-sncatransgenic mice were used to test the GM37, GM285 and comparator h9E4for their ability to block the alpha synuclein mediated effect.

Recordings were performed in all experiments 3 to 6 h followingadministration of a single dose of antibody at a dose of 15 mg/kg(i.p.). Basal synaptic transmission were recorded in both hippocampi ineach animal when possible, and recorded as individual experiments.

Acute treatment with h9E4 induced a significant reversal of theimpairment in basal synaptic transmission in F28-snca transgenic mice(Tg-snca+h9E4 vs. Tg-snca+PBS, p=0.002, FIG. 14 ). However, the reversalby h9E4 was only partial, as indicated by a significantlydifferentiation to basal synaptic transmission in littermates treatedwith PBS (p=0.007).

Acute treatment with GM37 induced a significant reversal of theimpairment in basal synaptic transmission in F28-snca transgenic mice(Tg-snca+GM37 vs. Tg-snca+PBS, p=0.004, FIG. 15 ). Basal synaptictransmission in GM37-treated transgenic mice was not significantlydifferent from basal synaptic transmission in PBS-treated littermates,indicating a full reversal of the impairment (FIG. 15 ).

GM285 also induced a significant reversal of the impairment in basalsynaptic transmission in F28-snca transgenic mice (FIG. 16 ). Basalsynaptic transmission in GM285-treated transgenic mice was notsignificantly different from basal synaptic transmission in PBS-treatedlittermates, indicating a full reversal of the impairment.

Example 8. Microdialysis to Assess Human Alpha-Synuclein in the Brain ofAwake Freely Moving Animals

The push-pull microdialysis method was used to assess the levels ofhuman alpha-synuclein in brain interstitial fluid (ISF). Mice weresingle-housed in controlled temperature (22±1.5° C.) and humidityconditions (55-65%) and kept on a 12:12 hour light/dark cycle (lights onat 06:00 h). Food and water were available ad libitum. The current studywas performed in the hippocampus of F28-snca transgenic mice (50-54weeks old). To enable microdialysis in the hippocampus, mice wereanaesthetized with isoflurane and an intracerebral guide cannula (CMA)was stereotaxically implanted into the brain, positioning themicrodialysis probe in the hippocampus (co-ordinates of probe tip: 3.1mm posterior and 2.8 mm lateral from bregma, and 1.3 mm relative duramater) according to the atlas of Paxinos and Franklin 2001. Anchorscrews and acrylic cement were used for the fixation of the guidecannulas. After implantation of the cannula mice were allowed to recoverfrom the surgery for 2-3 days before dialysis.

On the day of the experiment, a 2-mm, 1000 kDa cut-off CMA probe wasinserted through the guide cannula. A probe was connected to amicrodialysis peristaltic pump with two channels (MAB20; Microbiotech)and operated in push-pull mode. The inlet tubing of the microdialysisprobe was connected to a peristaltic pump perfusing the probe withartificial cerebrospinal fluid (aCSF; in mM: 147 NaCl, 2.7 KCl, 1.2CaCl₂), 0.85 MgCl₂). The peristaltic pump was also connected to theoutlet tubing in order to prevent perfusion fluid loss from the probe,by pulling the fluid through the tubing. As a perfusion buffer, 25%bovine albumin fraction V (Sigma) was diluted to 0.2% with artificialCSF on the day of use and filtered through a 0.1-μm membrane. The actualflow rate of the pump was determined without having the probe connected.The sample tubes were weighed before and after sampling for a given timeperiod and the flow rate was calculated. The pump was then set to aconstant flow of 1 μL/min. A 120 min sampling regimen was usedthroughout the experiment period. To avoid interference of tissuedamage, the experimental window was set from 14 to 48 hr after probeimplantation. 14-16 h after the start of the experiments, GM37, human9E4 or isotype control(anti-HEL) were injected i.p. at 15 mg/kg, and anadditional 6 samples (12 h of collection) were collected. The dialysateswere stored at −80° C. Concentration of human alpha synuclein wasdetermined by ELISA (Covance ELISA kit).

The average of the two-three basal values (4 h-6 h) prior to antibodytreatment was taken as baseline and set to 100% for each animal. Datawas evaluated using two-way analysis of variance (ANOVA) with repeatedmeasures to evaluate statistical relevance. The basal levels of humanalpha-synuclein in hippocampus were 8.1±1.1 ng/ml (mean±SEM, n=25, notcorrected for the in vitro dialysis probe recovery). The administrationof GM37 induced a larger reduction in human alpha-synuclein in thehippocampus of F28 mice compared to both the comparator antibody, human9E4, and the isotype control (anti-HEL). (FIG. 17 ).

Example 9: Chronic Effects of Alpha-Synuclein Antibodies In Vivo.Antibody GM37 Ameliorate Motor Phenotype in Rat Parkinson Model

Targeted overexpression of human alpha-synuclein to dopaminergic neuronsin the rat midbrain can be achieved using a recombinant adeno-associatedviral vector (rAAV) and is associated with a progressive loss ofdopaminergic cells in the substantia nigra as well as motor impairments.

Adult female Sprague-Dawley rats (225-250 g) were used to express humanalpha-synuclein in substantia nigra (SN) by injection with Adenoassociated virus of AAV2/5 serotype containing chicken beta-actinpromoter with enhancer elements from the cytomegalovirus promoter,followed by human alpha-synuclein cDNA and WPRE element as previouslydescribed (Xu L, Daly T, Gao C, Flotte T R, Song S, Byrne B J, Sands MS, Ponder K P (2001). In this model it has been shown that humanalpha-synuclein expression leads to neurodegeneration of dopaminergicneurons. Maingay M, et al. CNS Spectr. 2005 March; 10(3):235-44). Totest the effect of an alpha synuclein therapeutic antibody in this modelantibody treatment was initiated 2 to 4 days prior to viral injections,and continued until the end of the study (FIG. 18 ). PBS administrationat the same volume (5 ml/kg: IP) was used as a control. GM37 was dosedtwice per week at a dose of 15 mg/kg (IP). The viral particles (rAAV2/5)containing the gene for human wt alpha-synuclein or green fluorescentprotein (GFP) were injected unilaterally in the SN. Animals wereanaesthetized with a combination of Hypnorm® and Dormicum® at 2.0 ml/kgs.c. and placed in a stereotaxic frame. Their temperature was adjustedto 37.5° C. via a heating pad, and their skull was exposed. A hole wasdrilled above the right SN at the following coordinates, according tothe atlas of Paxinos and Watson (Paxinos & Watson, 1998): 5.5 mmposterior and 2.0 mm lateral from Bregma. A single injection of 3 μL ofrAAV2/5-alpha-syn or rAAV2/5-GFP was performed at a depth of 7.2 mmbelow the dura matter, and a flow rate of 0.2 μL/min using a Hamiltonsyringe connected to a stereotaxic injector. The needle was left inplace an additional 5 min to allow diffusion of the vector in the SN.Following surgery, the animals were returned to their home cage, andplaced in a heated environment where they were allowed to recover fromanesthesia. Testing of motor asymmetry in the cylinder test wasevaluated prior to AAV injections, as well as 3, 7 and 10 weeksfollowing AAV injections. The data presented correspond to the ratiobetween use of the right forepaw compared to the total use of both theleft and right forepaws. Each animal's performance in the cylinder wasfilmed for a total 5 min, and manual scoring of the number of touchesusing the left and right forepaws for 5 minutes has been performed forthe final testing day 10 weeks after virus injection. A significantimpairment is present in AAV-syn compared to AAV-GFP injected rats(p=0.012) at week 10. A trend for a reversal for GM37 treated animalswas shown, as their performance is different from GFP rats (p=0.163 andp=0.407 for gm37, respectively). This finding indicates that antibodyGM37 is able to ameliorate the Parkinsonian motor phenotype in this ratmodel (FIGS. 18 and 19 ).

Example 10: Chronic Effects of Alpha Synuclein Antibodies In Vivo.Antibody GM37 Inhibits Seeding of Endogenous Mouse Alpha SynucleinAggregation and Phosphorylation

Injection of alpha synuclein preformed fibrils made from recombinantprotein into dorsal striatum of wild type mice recruit endogenous mousealpha synuclein and induce formation of Ser-129 phosphorylatedaggregates inside neurons in cortex, amygdala and substantia nigra (Luket al. 2012, Science. 2012 Nov. 16; 338(6109):949-53). To see ifalpha-synuclein specific monoclonal antibody GM37 could reduce theappearance of alpha-synuclein fibril-induced phosphorylated alphasynuclein inclusion formation in vivo a total of 45 mice were used. Micewere dosed with GM 37 at 30 mg/kg i.p, GM 3715 mg/kg i.v., or vehicle ip(PBS). One day later the mice were anesthetized and stereotacticallyinjected in one hemisphere with 2 ul of recombinant human alpha-Syncrude seeds, made as described previously (Example 6) (total of 2 μgcrude seeds per animal). To inject the crude seeds, the skull was openedby boring a hole and a single glass pipette was inserted (co-ordinates:+0.5 mm anterior to Bregma, +2.0 mm lateral to midline) into the rightforebrain to target the inoculum to the dorsal neostriatum (+2.6 mmbeneath the dura). Following recovery, the mice received weekly i.p. ori.v. injections of antibodies until sacrifice at 45 days. Groups of 15mice/group were dosed either iv w. GM37 15 mg/kg, ip with GM37 30 mg/kg,or PBS (10 ml/kg) ip once weekly.

To measure the antibody concentration in plasma, cheek blood was drawnonce weekly just prior to next injection, ie 7 days after lastinjection. Plasma was obtained by a 2000 g spin, 15 min incubation atRT, supernatant was subsequently frozen at −20° C. A CSF sample wastaken at the end of the study and frozen at −20° C. Plasma and CSFsamples were analysed to determine the concentration of Human IgG byMSD. In short, mouse anti-human IgG (clone MH16-1 (M1268) was used forcapture, plasma or CSF was incubated in the well, followed by asulfo-TAG goat anti-human as the detection antibody (MSD cat no:R32AJ-1). Plates were analysed from electrochemiluminesence by MSD.

The antibody levels in plasma are shown in FIG. 20B and show a dosedependent increase in antibody plasma concentration and accumulation ofantibody in plasma during the six weeks. The antibody levels in csf areshown in FIG. 20C, and show that around 0.1% of antibody level in plasmacan be measured in csf.

At day 45, from the time of the injection of the alpha synucleinfibrillary seed, the mice were anesthetized, transcardially perfusedwith PBS, followed by perfusion with neutral buffered paraformaldehyde(4%). The brains were removed and incubated overnight for post-fixationin neutral buffered paraformaldehyde. Immunohistochemistry was performedon 45 μm thick serial sections by Neuroscience associates. Briefly,Using MultiBrain® technology, up to 25 mouse brains were embeddedtogether per block, into 3 blocks, freeze-sectioned at 45 μm thicknessin the coronal plane, and collected into cups containing antigenpreserve solution. Every sixth section was stained with antibody toSer-129 phosphorylated alpha-synuclein (Anti-alpha Synuclein (phosphoS129) antibody [Psyn/81A] ab184674) to reveal Seri 29 phosphorylatedalpha synuclein reactive structures.

Quantitation of pSyn pathology was performed by manual countingimmunoreactive positive cells from images at 10× magnification from 5-7sections covering the entire substantia nigra from every sixth section.The counting was performed blinded. Cell counts in amygdala and nigrawere analysed by a one-way ANOVA followed by Bonferoni t-test, where theeffect of GM37 antibody was compared to PBS treatment.

As can be seen from FIG. 20C, treatment with antibody GM37 reduced thenumber of intra-cellular inclusions in Substantia Nigra significantlywhen compared to PBS control, with either ip or iv treatment. The datashows that antibody GM37 could have therapeutic effect in PD by blockingentry of extracellular pathological alpha-synuclein into neurons, byblocking its propagation between neurons and/or facilitating clearancefrom the ISF by uptake into microglia. As this appearance of inclusionshas been linked to loss of dopaminergic neurons and development ofParkinsonian motor deficits in animal models, treatment with antibodyGM37 could have a therapeutic effect on loss of dopaminergic cells anddevelopment of motor deficits in PD.

Example 11: Manufactability of GM37 and GM37 Variants

The anti-alpha-synuclein antibodies are produced in mammalian cellculture under conditions that mimic the production conditions that willbe used for producing clinical grade material for use in patients. It iswell known that proteins produced in this manner undergopost-translational modifications that can impact both therapeuticpotency of the antibody as well as biophysical attributes that affectthe stability of the antibody over time. Empirical knowledge ascertainedfrom decades of studies identified a set of post-translationalmodifications known to provide risk for the developability of a specificmolecule. These post-translational modifications have been shown tocorrelate with amino acid strings present in the primary sequence of theheavy and light chain proteins. Algorithms have been generated that canidentify these sequences and determine the potential risk they will haveon the manufacturability and developability of a therapeutic antibody.

In silico analysis of the primary sequence of the antibody can be usedto de-risk a molecule for its potential to be developed as atherapeutic. In particular, detailed analysis of the VH and VL regionscan identified unique amino acids that are deemed important for themolecules activity but also may be a potential risk for its stabilityover time. Sequence specific deamidation has been identified as apotential risk for protein structures. Protein deamidation can occur onthe amide side chains of glutamines or asparagine residues and transformthem into a carboxylate group (Lorenzo et al. PLOSone, D01:10.1371,December (2015)). Nonenzymatic deamidation at neutral pH occurs fasterfor asparagine and is therefore considered a higher risk than glutamine.The activity is further influenced by the subsequent amino acid in thesequence and can occur at a rate of days or years. The actual fate ofthe protein that undergoes deamidation needs to be evaluatedexperimentally to determine the impact of the change both on itsstability and activity.

We identified a site for deamidation within the VH domain of GM37. Aminoacid residues 54 is an asparagine (N) followed by a glycine (G) atposition 55. The N54 is at high risk for spontaneous deamidation. Tomitigate this risk we generated a set of 3 variants that replace theasparagine (N) with serine(S), glutamine (Q) or histidine (H). All 3variants were produced in mammalian cell culture using transienttransfection methods (example 1.5). All 3 variants showed similarexpression and purification properties as GM37 wt. (FIG. 23 ).

For each of the eight products 400 ml transient transfections wereperformed using CHOK1SV GS-KO cells which had been in culture forminimum 2 weeks. Cells were sub-cultured 24 hours prior to transfection.All transfections were carried out via electroporation using Gene PulseXCell (Bio-Rad). For each transfection, viable cells were resuspended inpre-warmed CD-CHO media supplemented with 6 mM L-glutamine to 2.86×10⁷cells/ml. 40 μg of each established SGV DNA containing the appropriateheavy and light chains were aliquoted into each cuvette (Bio-Rad,GenePulser cuvette, 0.4 cm gap, 165-2088) and 700 μl cell suspensionadded. Cells were electroporated at 300V, 900 μF. Transfected cells weretransferred to rep-warmed media in Erlenmeyer flasks and the contents ofthe cuvettes rinsed twice with prewarmed media were also transferred tothe flasks. Transfectant cultures were incubated in a shaking incubatorat 36.5° C., 5% CO₂, 85% humidity, 140 rpm for 6 days. Cell viabilitywas measured at the time of harvest using a Cedex HiRes automated cellcounter (Rosche).

In order to evaluate the importance of residue 54 in binding to humanalpha-synuclein we analyzed the ability of the variants to bind in twodifferent experiments. Using a competition ELISA format we evaluated theimpact the change at residue 54 would have on the ability of GM37 tobind alpha-synuclein in solution. By evaluating the concentration ofsynuclein able to inhibit binding of the antibody to synuclein coatedELISA plates we showed all three variants maintained the same binding asGM37 wt and bind to alpha-synuclein with high affinity resulting inIC50s of 1-2 nM (FIG. 24 ). A competition assay was performed usingpreincubation of a fixed concentration (0.3 μg/ml) of each of thefollowing antibodies, GM37 (named GM 37 wt), GM37 variant1, GM37variant2 and GM37 variant3 with a range of 0-1000 nM humanalpha-synuclein for 60 minutes at room temperature. The remainingunbound antibody was captured and measured on ELISA plates coated with100 ng/ml of recombinant human alpha-synuclein using an anti-humandetection antibody by electrochemiluminesence (MSD, Gathersburg, Md.).The 1050s of the interaction are 1.9 nM, 1.6 nM, 2.1 nM and 1.4 nM forGM37 wt, GM37variant1, GM37variant2 and GM37variant3, respectively (asdetermined using Prism Graphpad®).

Using surface plasmin resonance (SPR), we evaluated the real timekinetics of binding of GM37 wt (2 batches) and the three variants(Example 2). The human alpha-synuclein was captured to the slide(ligand) and the antibodies were each tested at multiple concentrationsas analytes. Analysis of the binding curves in the presence of antibodyat multiple concentrations showed that the on rates were the same forall four antibodies, similarly when the antibody was removed from thebuffer the off-rates measured showed no statistical difference betweenthe antibodies. Using a 1:1 binding algorithm all 4 antibodies have nearidentical binding constants (FIG. 25 ).

In order to evaluate the impact of the changes at N54 on the functionalactivity of GM37 we analyzed the ability of the antibodies to blocksynuclein seeding activity in a culture of primary neurons (Example 6).The level of seeding is measured using an antibody specific forphospho-synuclein. All 3 antibodies were able to block seeding asmeasured by the phospho-synuclein signal (FIG. 26 ). Furthermore, thelevel of inhibition was the same for all 4 antibodies. This call baseddata further confirms the binding data that amino acid 54 in the VHdomain is not required for binding affinity to human alpha-synuclein orfor inhibition of seeding in a primary cell based assay. Furthermore, wefound that all three of these antibodies were capable of productionusing standard expression and purification methods. Interestingly one ofthe variants N54Q showed improvement in production over the othervariants, which is of great importance when the antibody is to beproduced commercially on large scale. These data support the possibilityof reducing the potential risk of deamidation by replacing asparagine(N) with another amino acid without concern over the loss of potency.

A samples of each of the antibodies wt GM37, var1, var2 and vara wassubjected to a steady increase in temperature over time and the level ofaggregation was simultaneously measured by multi-angle light scattering(Prometheus NT.48, NanoTemper Technologies). The temperature for onsetof aggregation was found to be similar for GM37 and the GM37-variants,however the lowest level of aggregation observed for GM37-Var2 (FIG. 27).

The invention claimed is:
 1. A method of diagnosing a synucleinopathy ina human subject, said method comprising: (a) administering to saidsubject a detectably-labeled antibody which binds to human alphasynuclein, wherein the antibody comprises: (i) heavy chain CDR1, CDR2,and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 21, and22, respectively, and light chain CDR1, CDR2, and CDR3 comprising theamino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively; (ii)heavy chain CDR1, CDR2, and CDR3 comprising the amino acid sequences ofSEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, andCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6,respectively; (iii) heavy chain CDR1, CDR2, and CDR3 comprising theamino acid sequences of SEQ ID NOs: 1, 33, and 3, respectively, andlight chain CDR1, CDR2, and CDR3 comprising the amino acid sequences ofSEQ ID NOs: 4, 5, and 6, respectively; (iv) heavy chain CDR1, CDR2, andCDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 34, and 3,respectively, and light chain CDR1, CDR2, and CDR3 comprising the aminoacid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or (v) heavychain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ IDNOs: 1, 35, and 3, respectively, and light chain CDR1, CDR2, and CDR3comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6,respectively, (b) measuring the binding of the antibody toalpha-synuclein in the subject, (c) comparing the level ofalpha-synuclein in the subject with a control level of alpha-synuclein,and (d) diagnosing the subject with a synucleinopathy if the level ofalpha-synuclein in the subject is increased relative to the controllevel of alpha-synuclein.
 2. The method of claim 1, wherein the bindingof the detectably-labeled antibody to alpha-synuclein is detected by invivo imaging.
 3. The method of claim 1, wherein the binding of thedetectably-labeled antibody to alpha-synuclein is detected by ex vivoimaging.
 4. The method of claim 1, wherein the antibody comprises: (a) aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO: 26 and a light chain variable domain comprising the amino acidsequence of SEQ ID NO: 27; (b) a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 7 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 8; (c) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequenceof SEQ ID NO: 8; (d) a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 31 and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO: 8; or (e) a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 32 anda light chain variable domain comprising the amino acid sequence of SEQID NO:
 8. 5. The method of claim 1, wherein the antibody is a singlechain Fv, a disulphide-bonded Fv, an Fab fragment, a Fab′ fragment, or aF(ab)₂ fragment.
 6. The method of claim 1, wherein the antibody is ahuman antibody.
 7. The method of claim 1, wherein the detectable labelis a fluorescent label, a chemiluminescent label, a paramagnetic label,a radioisotopic label, or an enzyme label.
 8. The method of claim 1,wherein the control level of alpha-synuclein corresponds to the level ofalpha-synuclein in a normal tissue sample.
 9. The method of claim 1,wherein the alpha-synuclein is detected in the brain or a body fluid ofthe subject.
 10. The method of claim 1, wherein the synucleinopathy isParkinson's disease.
 11. The method of claim 1, wherein thesynucleinopathy is selected from the group consisting of Gaucher'sDisease, Diffuse Lewy Body Disease, Lewy body variant of Alzheimer'sdisease, combined Alzheimer's and Parkinson's disease, pure autonomicfailure, and multiple system atrophy.
 12. A method of detecting humanalpha-synuclein in a human subject, the method comprising administeringto the subject a detectably-labeled antibody which binds to humanalpha-synuclein and detecting the binding of the antibody toalpha-synuclein, wherein the antibody comprises: (a) heavy chain CDR1,CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20,21, and 22, respectively, and light chain CDR1, CDR2, and CDR3comprising the amino acid sequences of SEQ ID NOs: 23, 24, and 25,respectively; (b) heavy chain CDR1, CDR2, and CDR3 comprising the aminoacid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and light chainCDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively; (c) heavy chain CDR1, CDR2, and CDR3comprising the amino acid sequences of SEQ ID NOs: 1, 33, and 3,respectively, and light chain CDR1, CDR2, and CDR3 comprising the aminoacid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (d) heavy chainCDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:1, 34, and 3, respectively, and light chain CDR1, CDR2, and CDR3comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6,respectively; or (e) heavy chain CDR1, CDR2, and CDR3 comprising theamino acid sequences of SEQ ID NOs: 1, 35, and 3, respectively, andlight chain CDR1, CDR2, and CDR3 comprising the amino acid sequences ofSEQ ID NOs: 4, 5, and 6, respectively.
 13. The method of claim 12,wherein the antibody comprises: (a) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 26 and a light chainvariable domain comprising the amino acid sequence of SEQ ID NO: 27; (b)a heavy chain variable domain comprising the amino acid sequence of SEQID NO: 7 and a light chain variable domain comprising the amino acidsequence of SEQ ID NO: 8; (c) a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 30 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 8; (d) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequenceof SEQ ID NO: 8; or (e) a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO: 32 and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:
 8. 14. The method ofclaim 12, wherein the antibody is a single chain Fv, a disulphide-bondedFv, an Fab fragment, a Fab′ fragment, or a F(ab)₂ fragment.
 15. Themethod of claim 12, wherein the antibody is a human antibody.
 16. Themethod of claim 12, wherein the detectable label is a fluorescent label,a chemiluminescent label, a paramagnetic label, a radioisotopic label,or an enzyme label.
 17. The method of claim 12, wherein thealpha-synuclein is detected in the brain or a body fluid of the subject.18. The method of claim 12, wherein the subject has Parkinson's disease.19. The method of claim 12, wherein the subject has a synucleinopathy.20. The method of claim 19, wherein the synucleinopathy is selected fromthe group consisting of Gaucher's Disease, Diffuse Lewy Body Disease,Lewy body variant of Alzheimer's disease, combined Alzheimer's andParkinson's disease, pure autonomic failure, and multiple systematrophy.